Neutral/ion reactor in adiabatic supersonic gas flow for ion mobility time-of-flight mass spectrometry

ABSTRACT

The content of the invention comprises a concept of reactor for isolated ion transformations induced by collisions with neutral species. This reactor is also an interface between mobility cell and orthogonal injection TOFMS based on supersonic adiabatic gas flow with variable controlled composition directed along the axis of a multipole ion guide with sectioned rods for possibility of creating of controlled distributions of RF, DC and AC rotating fields.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority to U.S. provisional application Ser.No. 60/817,338, filed on Jun. 29, 2006.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

This invention was made in part with government support. The UnitedStates Government has may have certain rights in the invention.

TECHNICAL FIELD

The present invention relates generally to instrumentation andmethodology for characterization of chemical samples based on ionmobility spectrometry (IMS) and mass spectrometry (MS).

BACKGROUND OF THE INVENTION

An ion mobility spectrometer typically comprises an ionization source, adrift cell, and an ion detector. Examples of an ion detector include afaraday sampling plate or cup, an electron multiplier, or a massspectrometer. Ion mobility spectrometry characterizes ions which areforced by an electric field to move a drift/buffer gas by measuring theion's equilibrium drift velocity. When gaseous ions in the presence ofthe drift gas experience a constant electric field, they accelerateuntil the occurrence of a collision with a neutral atom or moleculewithin the drift gas. This acceleration and collision sequence isrepeated continuously. Over time, this microscopic scenario averages theinstantaneous velocities over the macroscopic dimensions of the drifttube resulting in the measurement of a constant ion velocity based uponion size, charge and drift gas pressure. The ratio of the ion velocityto the magnitude of the electric field is defined as ion mobility. Inother words, the ion drift velocity (ν_(d)) is proportional to theelectric field strength (E), where the ion mobility K=ν_(d)/E is afunction of ion volume/charge ratio. Thus IMS is a separation techniquesimilar to mass spectrometry. IMS is generally known to have highsensitivity with moderate resolving power. Separation efficiency iscompromised when “bands” of ions spread apart as opposed to arrivingtogether at the end of the IM drift tube in a tight, well-definedspatial region.

The resolving power of an ion mobility cell increases as the square rootof the voltage when a uniform (or quasi-uniform) electric field isimposed across an ion mobility cell. It would seem that there is notmuch freedom to increase the resolution; however, the situation may beimproved if the ion drift in a gas flow is considered. Ions move againsta counterflow of gas only if the field is stronger than a certain valuespecific for the mobility of the ions. Ions with lower mobility may bestationary or even move in the negative direction (with the gas flow).Therefore, better ion separation can be expected where the time of themobility separation can be chosen suitable for specific applications andcompatible with the time diagram of the ion detector operation. Theproblem is how to efficiently organize ion mobility separation using gascounter-flow. Most often ion mobility separation is used with ionsources working under elevated pressure and the source pressure is oftenused when these ions are introduced into a mobility cell. There may beno gas counter-flow in such an application. On the other hand, drift gascounter-flow is inevitable when IMS is used for analysis of ions createdin high vacuum ion sources such as a secondary ion source wheresecondary ions are created from a surface maintained in high vacuum andmust then be moved against a counter-flow of gas into the ion mobilityspectrometer. The main problem then is how to overcome the strongcounter-flow and preserve ion throughput. It is quite natural to use atime varying electric field to gradually move ions from a pulsed ionformation region against the gas flow and into the IMS. Small ions needa relatively small field to overcome the gas flow without decomposingwhereas larger ions can come to the entrance orifice later under theaction of a stronger field. At the time of application of the largerfield necessary to move the heavier ions, small ions are already insidethe mobility cell and are not subjected to the strong field which wouldotherwise cause their fragmentation. Some separation of ions in additionto the usual mobility separation is achieved in this case, however, itis often rather small, because of the diffusion broadening during theinitial ion cloud formation.

The combination of an ion mobility spectrometer (IMS) with a massspectrometer (MS) is well known in the art. In 1961, Barnes et al. wereamong the first to combine these two separation methods. Suchinstruments allow for separation and analysis of ions according to boththeir mobility and mass, which is often referred to as two-dimensionalseparation or two-dimensional analysis. Young et al. realized that atime-of-flight mass spectrometer (TOFMS) and specifically an orthogonalTOFMS is the most preferred mass spectrometer type to be used in suchcombination because of its ability to detect simultaneously and veryrapidly (e.g. with high scan rate) all masses emerging from the mobilityspectrometer. The combination of a mobility spectrometer with a TOFMS isreferred to as a Mobility-TOFMS. This prior art instrument comprisedmeans for ion generation, a mobility drift cell, a TOFMS, and a smallorifice for ion transmission from the mobility cell to the TOFMS.

In 2003, Loboda (U.S. Pat. No. 6,630,662) described a method forimproving ion mobility separation by balancing ion drift motionsprovided by the influence of DC electric field and counter-flow of thegas. Using this balance, ions are at first accumulated inside an ionguide, preferably an RF-ion guide, and then, by changing the electricfield or gas flow, the ions are gradually eluted from the ion guide tothe mass spectrometer. Such type of ion accumulation is restricted tocollecting relatively small number of ions due to space-charge effects.It also has some limitation in ion mass-to-charge (m/z) range becauseRF-focusing for a given RF-voltage has decreasing efficiency for largermass ions which cannot be improved by increasing the RF-voltage due tothe possibility of creating a glow discharge at the relatively high gaspressure inside the RF multipole. Unfortunately at lower pressure theinfluence of the gas flow on ions is less and the diffusion of the ionsincrease so trapping and separation of larger ions could be compromised.The time of ion accumulation and their storage in the RF-ion guidecannot be too long, otherwise ions would be partially lost due todiffusion into rods or walls confining the gas flow. For at least thesereasons, this method has significant resolving power limitations. Theinstrumental improvements disclosed below eliminate these drawbacks.

Use of MS as a detector enables separation based on mass-to-charge (m/z)ratio after the separation based on ion mobility. Shoff and Hardenpioneered the use of Mobility-MS in a mode similar to tandem massspectrometry (MS/MS). In this mode, the mobility spectrometer is used toisolate a parent ion and the mass spectrometer is used for the analysisof fragment ions (also called daughter ions), which are produced byfragmentation of parent ions. Below this specific technique of operatinga Mobility-MS is referred to as Mobility/MS, or as Mobility-TOF if themass spectrometer is a TOFMS-type instrument. Other prior artinstruments and methods using sequential IMS/MS analysis have beendescribed (see, e.g., McKight, et al. Phys. Rev., 1967, 164, 62; Young,et al., J. Chem. Phys., 1970, 53, 4295; U.S. Pat. Nos. 5,905,258 and6,323,482 of Clemmer et al.; PCT WO 00/08456 of Guevremont) but nonecombine the instrumental improvements disclosed here. When coupled withsoft ionization techniques and the sensitivity improvements obtainedthrough the use of the drift cell systems disclosed herein, the IMS/MSsystems and corresponding analytical methods of the present inventionoffer significant analytical advantages over the prior art, particularlyfor the analysis of macromolecular species, such as biomolecules.

One challenge when building a Ion Mobility-MS system is to achieve highion transmission from the mobility region into the MS region. It is atthis interface that earlier uses of linear fields appear incongruouswith the goal of maximizing ion throughput across the IMS/MS interface.The mobility section operates at typical pressures between 1 mTorr and1000 Torr whereas the MS typically operates at pressures below 10⁻⁴Torr. In order to maintain this difference in pressure it is necessaryto restrict the cross-section of the exit orifice of the IM drift cellso that the region between the IM and the MS can be differentiallypumped. Typically this orifice cross section is well below 1 mm². Henceit is desirable to focus the ions into a narrow beam before they reachthe interface. Another essential property of the ion beam coming into anoTOFMS is the beam divergence, or the kinetic energy of ion motion inthe plane orthogonal to the direction of their travel. This is the mainfactor responsible for the quality of mass spectra obtained in theorthogonal TOFMS. It is a subject of our two co-pending U.S. patentapplications: U.S. application Ser. No. 11/441,766 filed May 26, 2006;and U.S. application Ser. No. 11/441,768 filed May 26, 2006 to achievegood ion beam properties by using ion cooling in supersonic adiabaticgas flow. Both of these applications are incorporated by reference asthough fully set out herein.

Tandem mass spectrometry techniques typically involve the detection ofions that have undergone some structural change(s) in a massspectrometer. Frequently, this change involves dissociating orfragmenting a selected precursor or parent ion and recording the massspectrum of the resultant daughter fragment ions. The information in thefragment ion mass spectrum is often a useful aid in elucidating thestructure of the precursor or parent ion. The general approach used toobtain a mass spectrometry/mass spectrometry (MS/MS or MS²) spectrum isto isolate a selected precursor or parent ion with a suitable m/zanalyzer, subject the precursor or parent ion to an energy source toeffect the dissociation (e.g. energetic collisions with a neutral gas inorder to induce dissociation), and finally to mass analyze the fragmentor daughter ions in order to generate a mass spectrum. An additionalstage of isolation, fragmentation and mass analysis can be applied tothe MS/MS scheme outlined above, giving MS/MS/MS or MS³. This additionalstage can be quite useful to elucidate dissociation pathways,particularly if the MS² spectrum is very rich in fragment ion peaks oris dominated by primary fragment ions with little structuralinformation. MS³ offers the opportunity to break down the primaryfragment ions and generate additional or secondary fragment ions thatoften yield the information of interest. The technique can be carriedout n times to provide an MS^(n) spectrum.

Ions are typically fragmented or dissociated in some form of a collisioncell where the ions are caused to collide with an inert gas.Dissociation is induced usually either because the ions are injectedinto the cell with a high axial energy or by application of an externalexcitation. See, for example, WIPO publication WO 00/33350 dated Jun. 8,2000 by Douglas et al. Douglas discloses a triple quadrupole massspectrometer wherein the middle quadrupole is configured as a relativelyhigh pressure collision cell in which ions are trapped. This offers theopportunity to both isolate and fragment a chosen ion using resonantexcitation techniques. The problem with the Douglas system is that theability to isolate and fragment a specific ion within the collision cellis relatively low. To compensate for this, Douglas uses the firstquadrupole as a mass filter to provide high resolution in the selectionof precursor ions, which enables an MS² spectrum to be recorded withrelatively high accuracy. However, to produce an MS³ (or higher)spectrum, isolation and fragmentation must be carried out in thelimited-resolution collision cell.

A three-dimensional ion trap (3-D IT) is one of the most flexibledevices for MS-MS and multi-step (MS^(n)) analysis. The basic operationprinciple of the quadrupole ion trap mass spectrometer is well-known(for example, refer to U.S. Pat. No. 2,939,952, Paul et al., June,1960). This trap is composed of a ring electrode and two end capelectrodes of special shape to create a quadrupolar distribution ofpotential. Radio frequency (RF) and DC offset electric potentials areapplied between electrodes and cause ions to oscillate within the trap.By appropriately selecting voltage parameters, ions of a specificmass/charge ratio can be made to have stable or unstable trajectories.In another implementation an additional (auxiliary) AC voltage isapplied to the end-caps to induce resonant excitation of selected ionseither for the purpose of ejecting the selected ions or for the purposeof inducing collisional dissociation. The 3-D ion trap is capable ofsingle step mass spectrometric analysis. In such analysis ions areinjected into the trap (or generated within the trap), confined to thecenter of trap because of low energy collisions with an inert gas suchas helium (typically at 1 mtorr pressure) and then sequentially ejectedthrough the apertures in the end cap electrodes onto an externaldetector by raising the amplitude of the RF field. The same device couldbe used for a multi-step, i.e. MS^(n), analysis—U.S. reissued Pat. No.34,000, Syka et al., July, 1992. The ion trap isolates ions in a m/zwindow by rejecting other components, then fragments these isolated ionsby AC excitation, then isolates resulting ion fragments in a m/z windowand repeats such sequence (MS^(n) operation) in a single cell. At theend of the sequence ions are resonantly ejected to acquire the massspectrum of N-th generation fragments. The 3-D IT is vulnerable tosensitivity losses due to ion rejection and instability losses at thetime of ion selection and fragmentation.

Another version of this technique is provided by hybrid instrumentscombining quadrupoles with time of flight analyzers (Q-TOF) where thesecond quadrupole mass spectrometer (in a triple quadrupole systems) isreplaced by an orthogonal time of flight spectrometer (o-TOF). The o-TOFback end allows observation of all fragment ions at once and theacquisition of secondary spectra at high resolution and mass accuracy.The Q-TOF has huge advantages in cases where the full mass range ofdaughter ions is required, for example, for peptide sequencing, theQ-TOF strongly surpasses the performance of the triple quadrupole.However, the Q-TOF suffers a 10 to a 100 fold loss in sensitivity ascompared to a single quadrupole mass filter operating in selectedreaction monitoring mode (monitoring single m/z). For the same reasonthe sensitivity of the Q-TOF is lower in the mode of “parent scan”where, again, the second MS is used to monitor a single m/z. Usually,only one step of MS/MS analysis is possible for such types ofinstruments. Recently, the quadrupole has been replaced by a linear iontrap (LIT)—U.S. Pat. No. 6,020,586, Dresch et al., February, 2000. Thequadrupole with electrostatic “plugs” is capable of trapping ions forlong periods of time. The quadrupole field structure allows one to applyan arsenal of separation and excitation methods, developed in 3-D iontrap technology, combined with easy introduction and ejection of the ionbeam out of the LIT. The LIT eliminates ion losses at selection and alsocan operate at poor vacuum conditions which reduces requirements on thepumping system. However, a limited resolution of ion selection, R<200,has been demonstrated thus far. A method for improving the sensitivityin LIT is described in U.S. Pat. No. 6,507,019, Chernushevich et al.,January, 2003. According to this method, a voltage on the outlet of thecollision chamber is controlled in synchronization with the timing ofapplying an acceleration voltage in a time-of-flight mass spectrometerthereby improving the sensitivity for ions in a specified range of m/z.

Fourier transform ion cyclotron resonance mass spectrometry (FTMS)currently provides the most accurate measurement of ion mass to chargeratios with a demonstrated resolution in excess of 100,000. In FTMS,ions are either injected from outside the cell or created inside thecell and confined in the cell by a combination of static magnetic andelectric fields (Penning trap). The static magnetic and electric fielddefine the mass dependent frequency of cyclotron motion. This motion isexcited by an oscillating electric potential. After a short period theapplied field is turned off. Amplifying and recording weak voltagesinduced on the cell plates by the ion's motion detects the frequency ofion motion and, thus, the m/z of the ion. Ions are selectively isolatedor dissociated by varying the magnitude and frequency of the appliedtransverse RF electric potential and the background neutral gaspressure. Repeated sequences of ion isolation and fragmentation (MS^(n)operation) can be performed in a single cell. The possibility of kineticmeasurements of ion dissociation using controlled black body heating ofions so called BIRD technique is another unique property of these typeinstruments. However, it may be considered as rather basic research toolthan analytically useful approach. An FTMS is a “bulky” device occupyinga large footprint and is also expensive due to the costs of the magneticfield. Moreover, an FTMS exhibits poor ion retention in MS^(n) operation(relative to the 3-D ion trap).

Use of an AC field for selected ion rotation within a gas filled RFQaxis has been described (Raznikov, et. al., RCM, 15, 1912-1921, 2001).Such ion motion was used for ion heating and fragmentation of selectedions by collisions with buffer gas. It was demonstrated that resolvingpower (FWHM) was near 80 for mass selecting the parent ions (within them/z range 500-1000) which could then be selectively decomposed under N₂pressure close to 20 mTorr. Kinetic measurements are enhanced when usingthis technique (Soulimenkov, et. al., Europian Journal of MassSpectrometry 8, 99-105, 2002). Rotating ions around the axis of a gasfilled RFQ is one of the particular cases of two-dimensional motion inan axially symmetric quadratic potential well provided by a quadrupolarRF-field. This motion is influenced by harmonic voltages applied toadjacent RFQ rods with phase shift π/2. Upon comparison with other typesof ion oscillations in which the ion distance from RFQ axis varies overa wide range, ion rotation has some advantages conferred by theproperties of classic harmonic motion. For simple ion oscillations (fordipole or quadrupole excitations), the ion velocity is not constant andthe ions come to rest at the maximum deviation from the axis, whereasfor rotating ion motion the velocity is almost constant. There are twoadvantages to having a constant average kinetic energy for a givenmaximum rotational orbit deviation from the axis. First, as aconsequence of the constant ion velocity the conditions for observationof fragment ions, especially including low m/z values, can beconsiderably better known and controlled than merely using a quadrupolarfield alone. Otherwise, in order to achieve the same average ioninternal energy in the case of quadrupolar oscillations, it is necessaryto have either larger maximum deviations of ions from the RFQ axis or tohave a stronger ion focusing to the axis which then demands largeramplitude or lower frequency of the RF-voltage. The second advantage isthat ion rotation gives more control of ion heating and decompositionunder the usual conditions employed in the RFQ where the gas density isnearly uniform.

In addition to ICR mass spectrometry, other applications of ion rotationare found in DC traps like the Orbitrap instrument (disclosed by Makarovin 1999, U.S. Pat. No. 5,886,346) and FTMS based on ion rotation in alinear RF multipole ion trap (described by Park in 2004, U.S. Pat. No.6,784,421). In all these instruments ions rotate freely in high vacuumafter a short voltage pulse starts the ion motion. The linear RFmultipole ion trap is the closest prior art to ion rotation in a gasfilled RFQ. In both types of instrument the resolving power restrictionsare dependent on the accuracy of manufacturing of RF ion guide or lineartrap. The Orbitrap arguably has some important advantages over thesetechniques, however, it is limited to analyzing ions of only one signwhile both ICR and ion rotation in linear multipole ion traps allowsimultaneous measurement of ions of both signs. All these techniques mayprovide extremely high resolution for moderate to small ions. For largerions mass resolving power decreases rapidly for two main reasons: largeions rotate at a lower frequency than smaller ions and larger m/z aremore prone to oscillations and thus have shorter time periods ofuninterrupted coherent motion due to the increased probability of impactwith residual gas resulting from their larger collision cross section.Moreover, inserting large ions into the DC trap portion of the Orbitraphas a further limitation since these ions may have a large probabilityto collide with gas in the storage quadrupole during their accelerationto the necessary relatively high energy for insertion (about 1 kV).Thus, during insertion of these larger ions into the Orbitrap, asignificant portion of the large ion flux may be lost due todissociation broadening of their energy distribution away from anoptimal insertion energy at the moment of their capture in the DC trap.These instruments demand long measurement times (up to few seconds) andslightly more time for ion preliminary accumulation. Thus, they are notsuitable for many types of ion mobility measurements since all mobilityresolution will be lost during such ion accumulation as will anyinformation about the average velocity of the incoming ions. Also, mostoften mass spectrometric techniques provide only semi-quantitativeinformation about ion transformations. For real kinetic measurements itis necessary to have very narrowly defined energy distributions (ortemperatures) of ions and of neutral reactant components combined withprecise measurement of reaction times. The only methods capable ofmanipulating these parameters use a direct heating of the reaction zone(like BIRD, for example). However, external heating of some parts of theinstrument may result in significant experimental problems. The methodof selective ion heating with a resonant rotating field also has alimited capability to specify both the temperature of the ions ofinterest and the time at which heating occurs since the temperature ofrotating ions for a given field strength is dependent on ion mobility.Unfortunately, it is necessary to know this temperature under conditionswhen ions begin to decompose yet this temperature can only beeffectively measured when ions have not yet decomposed. Furthermore, tohave high selectivity with resonant ion rotation the buffer gas pressureshould be small which then requires an inordinately long time for theselected ions to reach the desired steady state temperature. The samelimitation occurs at the end of an RFQ when the rotating field isswitched off to allow focusing the ions for subsequent measurement inthe TOFMS.

Two dimensional, 2-D PAGE polyacrylamide gel electrophoresis is apopular and currently preferable technique for protein separation(Anderson N. G., Anderson N. L., Electrophoresis 1996; 17, 443453).Proteins are subjected at first to isoelectric focusing (IEF) in animmobilized pH gradient in the gel plate to separate proteins accordingto their charging abilities (pI values), a step which typically takesabout 6-8 hours. Then, the IEF gel is placed on top of a gradient geland is subjected to electrophoretic separation in the presence of SDS(sodium dodecyl sulphate). In SDS-PAGE, proteins are denatured anddissolved in a SDS buffer, negatively charged SDS molecules bind to theprotein, with more molecules binding to larger proteins. On applicationof an electric field, proteins migrate in a polyactylamide gel accordingto their charge connected with size or mass. The electric field isswitched off to immobilize the proteins within the gel. The separatedproteins are stained for visualization, bands of interest are excisedand digested with protease followed by mass spectrometry measurementsfor protein identification (Shevchenko, A., Jensen, O. N.,Podtelejnikov, A. V., Sagliocco, F., Wilm, M., Vorm, O., Mortensen, P.,Boucherie, H., Mann, M., Proc. Natl. Acad. Sci. U.S.A. 1996, 93,14440-14445; Jensen, O. N., Larsen, M. R., Roepstorff, P., PROTEINS1998, 74-89 Suppl. 2). 2-D PAGE is the current technology of choice forlarge scale proteomics analysis because 2-D PAGE at the moment is thehighest resolution method for protein separation and the spots ofproteins in the 2-D map are related to the properties of proteins,namely isoelectric point in the first dimension and the molecular massin the second dimension. Therefore, the positions of proteins in 2-D mapcorrespond to their chemical and physical properties. These propertiescan be used to identify and characterize the proteins. 2-D PAGE has beenused to analyze human plasma proteins, and the pI and molecular weightof proteins can be used for detection and diagnosis of diseases inclinical analysis (Rasmussen, R K., Ji, H., Eddes, J. S., Moritz, R L.,Reid, G. E., Simpson, R. J., Dorow, D. S., Electrophoresis 1997, 18,588-598). However, 2-D PAGE is a time consuming procedure which isdifficult to automate, it also suffers from limitations in sensitivityand dynamic range of detection. Virtual 2-D gel electrophoresis hasrecently been developed (Ogorzalek-Loo, R. R., Cavalcoli, J. D.,VaiBogelen, R A., Mitchell, C., Loo, J. A., Moldover, B., Andrews, P.C., Anal. Chem. 2001, 73, 40634070), where mass spectrometry replacesthe size-based separation of SDS-PAGE in the second dimension. It hasbeen shown that this technology is more sensitive than 2-D PAGE.However, the first dimension of separation is still performed in apolyacrylamide gel, limiting the potential for high throughput analysis.Capillary isoelectric focusing (CIEF) is an equilibrium-based method ofseparation that depends on a pH gradient created by carrier ampholyte.Proteins move under an electric field to their pI points where theycarry zero average charge and are focused. Therefore, separation andconcentration occur at the same time. The concentration of proteins atthe focused zone can be increased by 100-500 times relative to thestarting solution because the same protein in the whole capillary isfocused on a single spot. Single point detection techniques, such aslaser induced fluorescence and ESI-MS, have been employed to detect theseparated proteins after CIEF. Focused protein zones need to bemobilized in order to pass through the detection point at the end of thetube (Rodriguez, R., Zhu, M., Wehr, T., J Chromatogr. A 1997, 772,145-160). The problem of interfacing CIEF with MALDI-MS is also becausethe focused protein zone inside the capillary cannot be reacheddirectly. Therefore, the contents of the capillary need to be moved outof the capillary and deposited into an appropriate surface forsubsequent MALDI-MS analysis. This mobilization step degrades theresolution, increases the analysis time, and distorts the pH gradient.Hence, the result reproducibility is poor.

All of the above-referenced U.S. patents and published U.S. patentapplications are incorporated by reference as though fully describedherein.

Although much of the prior art has resulted in improvements in ionfocusing, separation and in ion throughput from ion source to themobility cell (and to the mass spectrometer in tandem instruments),there is room for additional improvement in all these areas. Theinventors describe herein a concept and design of a new type ofinterface of an ion mobility cell with an orthogonal injectiontime-of-flight mass spectrometer (TOFMS) based on significant cooling ofthe ion beam in an adiabatic supersonic gas flow and its focusing byradio-frequency quadrupole or multipole ion guide with additional DC andAC rotating fields which result in variety of instrumental embodimentsto provide improved ion production from investigated samples, theirseparation and measurements. The modified ion rotational trapping,manipulation, and measurement technique disclosed in the presentinvention is free from the limitations of the prior art as the mobilitycross-sections of any ion of interest will have always been measuredprior to its introduction into the RFM. Furthermore, the driftvelocities of these ions and those for the components of the buffer gasand their divergences (which can be related to their temperatures) areessential for obtaining quantitative kinetic information and theseproperties can be uniquely measured by innovative use of multianodeposition sensitive detector combined with a multi-channel data recordingsystem in a TOFMS. One of the aims of the present invention is toperform isoelectric separation of biomolecules in the gas phase andreduce the time of conventional procedure in gel separation from 6-8hours to few seconds or minutes depending on the problem to be solved.Also the problems of interfacing of TOFMS with separating devices inliquid phase would be avoided in this case.

Time-of-flight instruments seem to be more suitable than prior artinstruments for measurement of ions coming from an RF ion guide withsupersonic gas flow, especially when investigation of large bioions isthe main analytical problem. Divergence of large ions in the supersonicgas flow and their final focusing in an RF ion guide technically can bedone significantly better than those for relatively small ions.Therefore, it is quite realistic to provide better resolving power forlarge ions in TOF measurements than for those ions with less mass andsmaller size. Furthermore, the resolution of multicharged ions variesproportionally to the square root of the charge number at least for alinear oTOFMS. Moreover, the expected peak shape, at least for a lineargridless TOFMS, would be close to Gaussian which is significantly betterthen Lorentzian peak shapes typical for FTMS instruments. This is anespecially useful advantage when measuring a small peak adjacent to alarge one. Multianode data acquisition allows not only to increase thedynamic range of the ion detection but also to measure ion velocitiesand their divergences thus providing direct estimations of theirtemperatures. These possibilities are important for quantitative kineticmeasurements.

BRIEF SUMMARY OF THE INVENTION

The present invention is directed to a system and method forcharacterization of chemical samples based on ion mobility spectrometry(IMS) and mass spectrometry (MS).

In one embodiment of the present invention, there is an apparatus forthe analysis of gaseous ions and neutral species and mixtures thereof,the apparatus comprising: an ion source for the production of gaseousions and neutral species and mixtures thereof, a gas flow formationregion fluidly coupled to the ion source, the gas flow formation regioncomprising a sectioned capillary having at least two electrodes, the gasflow formation region operable to accept ions and/or neutral speciesfrom the ion source; at least one sectioned radio-frequency multipoleion guide fluidly coupled to the gas flow formation region, thesectioned ion guide comprising a plurality of electrically isolatedelectrode sections; an exit orifice fluidly coupled to the at least onesectioned radio-frequency multipole ion guide; and, a detector fluidlycoupled to the exit orifice. In some embodiments, the gas flow formationregion comprises an input orifice. In some embodiments, the inputorifice is coupled to a gas or liquid delivery valve. In someembodiments, the sectioned radio-frequency multipole ion guide comprisesmulitelectrode rods. In some embodiments wherein the sectionedradio-frequency multipole ion guide comprises multielectrode rods, themulitelectrode rods are separated by grounded plates isolated fromground. In some embodiments, the at least one sectioned radio-frequencymultipole ion guide comprises a radio-frequency quadrupole. In someembodiments, the at least one sectioned radio-frequency multipole ionguide comprises a radio-frequency octapole. In some embodiments, the atleast one sectioned radio-frequency multipole ion guide comprises adifferential pumping region. In some embodiments, the electricallyisolated electrode sections are coupled to a component selected from thegroup consisting of a DC voltage source, an AC voltage source, a RFvoltage source, and any combination thereof. In some embodiments whereinthe electrically isolated electrode sections are coupled to a componentselected from the group consisting of a DC voltage source, an AC voltagesource, a RF voltage source, and any combination thereof, the voltagesources are coupled to, and controlled by, a computer. In someembodiments, the gas flow formation region is a supersonic gas flowformation region. In some embodiments, the detector is a massspectrometer. In some embodiments wherein the detector is a massspectrometer, the mass spectrometer is an orthogonal time-of-flight massspectrometer. In some embodiments wherein the detector is a massspectrometer and the mass spectrometer is an orthogonal time-of-flightmass spectrometer, the orthogonal time-of-flight mass spectrometercomprises a position sensitive multi-anode detector. In some embodimentswherein the detector is a mass spectrometer and the mass spectrometer isan orthogonal time-of-flight mass spectrometer, the orthogonaltime-of-flight mass spectrometer is a bipolar time-of-flight massspectrometer. In some embodiments, the gas flow formation regioncomprises an exit capillary of the ion source. In some embodimentswherein the gas flow formation region comprises an exit capillary of theion source, the exit capillary comprises individually biasable gas-tightelectrodes. In some embodiments wherein the exit capillary comprisesindividually biasable gas-tight electrodes, the at least one electrodeof the exit capillary comprises a surface coated with dielectric films.In some embodiments, the at least one sectioned radio-frequencymultipole ion guide comprise a sectioned radio-frequency octopole ionguide followed by a sectioned radio frequency quadrupole ion guide. Insome embodiments, the ion source comprises an ion mobility cell. In someembodiments, the apparatus further comprises an electron source fluidlycoupled to the exit orifice and to the detector. In some embodimentswherein the apparatus further comprises an electron source fluidlycoupled to the exit orifice and to the detector, the electron source isa pulsed electron source, a continuous electron source, or a combinationthereof. In some embodiments, the apparatus further comprises a mirrorassembly between the exit orifice and the detector. In some embodimentswherein the apparatus further comprises a mirror assembly between theexit orifice and the detector, the mirror assembly comprises a parabolicmirror or a cylindrical mirror; and, a flat mirror.

In one embodiment of the present invention, there is a method ofanalyzing gaseous ions, neutral species or mixtures of ions and neutralspecies comprising: introducing the ions and/or neutral species into agas flow formation region to form a gas flow of the ions and/or neutralspecies, the gas flow formation region comprising a sectioned capillaryhaving at least two electrodes; introducing the gas flow of ions and/orneutral species into at least one sectioned radio-frequency multipoleion guide, the sectioned ion guide comprising a plurality ofelectrically isolated electrode sections; applying voltage selected fromthe group consisting of DC voltages, AC voltage, RF voltages, and anycombination thereof, to one or more sections of the at least onesectioned radio-frequency multipole ion guide; detecting the gas flow ofions and/or neutral species. In some embodiments, the method furthercomprises the step of varying one or more of the voltage selected fromthe group consisting of DC voltages, AC voltage, RF voltages, and anycombination thereof. In some embodiments, the step of applying voltageto the ion guide comprises producing a resonant rotating field whichextracts specific ions from the gas flow and causes the specific ions tofollow a rotational orbit around the central axis of the multipole ionguide. In some embodiments wherein the step of applying voltage to theion guide comprises producing a resonant rotating field which extractsspecific ions from the gas flow and causes the specific ions to follow arotational orbit around the central axis of the multipole ion guide, theresonant rotating field voltages are formed by applying an alternatingcurrent harmonic voltage with a phase shift of π/2 between adjacent rodswithin said radio-frequency multipole ion guide. In some embodimentswherein the step of applying voltage to the ion guide comprisesproducing a resonant rotating field which extracts specific ions fromthe gas flow and causes the specific ions to follow a rotational orbitaround the central axis of the multipole ion guide, the step of applyingvoltage to said ion guide further comprises producing a gradient DCfield which traps the ions following a rotational orbit in a region ofthe ion guide. In some embodiments wherein the step of applying voltageto said ion guide further comprises producing a gradient DC field whichtraps the ions following a rotational orbit in a region of the ionguide, the step of applying voltage extracts and traps ions of one ormore m/z values and ion mobility cross-sections. In some embodimentswherein the step of applying voltage to the ion guide comprisesproducing a resonant rotating field which extracts specific ions fromthe gas flow and causes the specific ions to follow a rotational orbitaround the central axis of the multipole ion guide, the resonantrotating field voltage is a graded resonant rotating field voltage. Insome embodiments, the step of applying voltage to the ion guidecomprises holding specific ions in the gas flow. In some embodiments,the step of introducing the ions and/or neutral species into a gas flowformation region comprises introducing the ions and/or neutral speciesinto a supersonic gas flow. In some embodiments wherein the step ofintroducing the ions and/or neutral species into a gas flow formationregion comprises introducing the ions and/or neutral species into asupersonic gas flow, the step of applying voltage to the ion guidecomprises trapping ions in the supersonic gas flow. In some embodimentswherein the step of applying voltage to the ion guide comprises trappingions in the supersonic gas flow, the step of introducing said ionsand/or neutral species into a gas flow formation region comprises addingan admixture gas. In some embodiments having an admixture gas, theadmixture gas comprises Ar and/or Xe. In some embodiments having anadmixture gas, the step of adding an admixture gas fragments the trappedions to create daughter ions of the trapped ions. In some embodimentswherein the step of adding an admixture gas fragments the trapped ionsto create daughter ions of the trapped ions, the step of applyingvoltage selected from the group consisting of DC voltages, AC voltage,RF voltages, and any combination thereof, to one or more sections ofsaid at least one sectioned radio-frequency multipole ion guide,extracts daughter ions from the gas stream. In some embodiments of themethod wherein daughter ions are extracted from the gas stream, themethod further comprises varying the voltage selected from the groupconsisting of DC voltages, AC voltage, RF voltages, and any combinationthereof to reintroduce the daughter ions into the gas stream. In someembodiments, the method further comprises applying a DC voltage betweenthe last electrode of the sectioned capillary and the first electrodesection of the radio-frequency multipole ion guide. In some embodimentswherein the method further comprises applying a DC voltage between thelast electrode of the sectioned capillary and the first electrodesection of the radio-frequency multipole ion guide, the DC voltagebetween the last electrode of the sectioned capillary and the firstelectrode section of the radio-frequency multipole ion guide is appliedwhen a desired ion is present in a region between the sectionedcapillary and the radio-frequency multipole ion guide. In someembodiments wherein the DC voltage between the last electrode of thesectioned capillary and the first electrode section of theradio-frequency multipole ion guide is applied when a desired ion ispresent in a region between the sectioned capillary and theradio-frequency multipole ion guide, the method further comprises thestep of removing the DC voltage between the last electrode of thesectioned capillary and the first electrode section of theradio-frequency multipole ion guide. In some embodiments wherein themethod further comprises the step of removing the DC voltage between thelast electrode of the sectioned capillary and the first electrodesection of the radio-frequency multipole ion guide, the steps ofapplying and removing the DC voltage between the last electrode of thesectioned capillary and the first electrode section of theradio-frequency multipole ion guide are repeated one or more times. Insome embodiments wherein daughter ions are extracted from the gasstream, the method further comprises the step of removing the voltageselected from the group consisting of DC voltages, AC voltage, RFvoltages, and any combination thereof, to one or more sections of the atleast one sectioned radio-frequency multipole ion guide. In someembodiments, the method further comprises the steps of applying adecreasing electric field in the direction of the gas flow in theradio-frequency multipole ion guide and measuring the mobilitycross-section of the ions In some embodiments of the method, the step ofdetecting comprises detecting with a mass spectrometer. In someembodiments wherein the step of detecting comprises detecting with amass spectrometer, the mass spectrometer is an orthogonal time-of-flightmass spectrometer. In some embodiments wherein the step of detectinguses an orthogonal time-of-flight mass spectrometer, the orthogonaltime-of-flight mass spectrometer comprises a position sensitivemulti-anode detector. In some embodiments wherein the step of detectinguses an orthogonal time-of-flight mass spectrometer, the orthogonaltime-of-flight mass spectrometer is a bipolar time-of-flight massspectrometer. In some embodiments, the method further comprises the stepof passing the gas flow of ions and/or neutral species into a mirrorassembly. In some embodiments of the method, the mirror assemblycomprises a parabolic mirror or a cylindrical mirror; and, a flatmirror. In some embodiments, the method further comprises the step ofimpacting the gas flow of ions and/or neutral species with electronsfrom an electron source or with photons from a laser source.

The foregoing has outlined rather broadly the features and technicaladvantages of the present invention in order that the detaileddescription of the invention that follows may be better understood.Additional features and advantages of the invention will be describedhereinafter which form the subject of the claims of the invention. Itshould be appreciated by those skilled in the art that the conceptionand specific embodiment disclosed may be readily utilized as a basis formodifying or designing other structures for carrying out the samepurposes of the present invention. It should also be realized by thoseskilled in the art that such equivalent constructions do not depart fromthe spirit and scope of the invention as set forth in the appendedclaims. The novel features which are believed to be characteristic ofthe invention, both as to its organization and method of operation,together with further objects and advantages will be better understoodfrom the following description when considered in connection with theaccompanying figures. It is to be expressly understood, however, thateach of the figures is provided for the purpose of illustration anddescription only and is not intended as a definition of the limits ofthe present invention.

BRIEF DESCRIPTION OF THE DRAWINGS

For a more complete understanding of the present invention, reference isnow made to the following descriptions taken in conjunction with theaccompanying drawing, in which:

FIG. 1. Schematic diagram of the proposed RFQ interface between amobility cell and an oTOFMS. Methods of trapping of ions with givenvalue of mobility and their heating by the flow of heavier gas atoms areillustrated. Formation of ion clusters in supersonic gas flow and theircontrolled decomposition are shown too.

FIG. 2. Cross section of RFQ-ion guide (from FIG. 1) showing the appliedfocusing and rotation voltages. Excitation of rotational motion ofselected taken ions from those trapped in the gas flow is shown. Therotational field may be used as for removing of undesired ions oralternatively for isolation of chosen ions for further transformations.

FIG. 3. Illustration of parent ion decomposition while removing productions from the zone of decomposition and further isolating and purifyingthese desired product ions with resonant rotational fields.

FIG. 4. Cross-section of one the sections of the RF multipole ion guidefor one beam of a multibeam IM TOFMS showing the applied RF focusing androtating fields for isolating of desired product ions from the firstdecomposition of isolated parent ions.

FIG. 5. Illustration of trapping of desired ions with chosen m/z valuefrom multiple mobility separations by use of rotating and DC fields.

FIG. 6. Illustration of trapping of desired ions with chosen m/z valueand mobility interval from multiple mobility separations by use ofrotating and DC fields.

FIG. 7. One possible way of measuring of ion mobility crossection ofions isolated at low temperature conditions as shown in FIGS. 5 and 6.Also shown is an electron impact ionizer which can be switched toprovide low energy electron capture dissociation of large multichargedpositive ions.

FIG. 8. Cross section view of combined ion guide with RF-octapole forthe trapping and isolating of ions and RF-quadrupole for focusing ionsand controlling their transformations under low temperature conditions.

FIG. 9. Illustrates the view along of the combined ion guide of FIG. 8.

FIG. 10. Schematic view of linear TOFMS with multichannel data recordingshowing the possibility of measuring of ion drift velocities anddivergence of the ion beams and distinguishing the ions formed byelectron impact inside the gas flow after exiting the RFQ ion guide fromthe product ions released from within the RFQ after switching off theretarding trapping potential.

FIG. 11. Schematic view of possible processes of proton exchange fordifferent sites of a biomolecule.

FIG. 12. Qualitative view of density distributions for differentcomponents of the gas flow at a cross section of the RFM at somedistance along the RFM after the IM exit tube.

FIG. 13. Illustration of selection of desired biomolecules by providingconditions close to their isoelectric point.

FIG. 14. Schematic view of a linear Bipolar Time-Of-Flight MassSpectrometer (BiTOFMS) and coincident recording of zwitter-ionsfragments produced by laser-induced dissociation.

FIG. 15. Schematic view of ion mirrors for transformation of divergentbipolar ion flow into quasi-parallel beam followed by its insertion intoa linear BiTOFMS.

FIG. 16. Schematic view of coupling of two adjacent supersonic gas flowRFQ ion guides in series showing independent trapping in the second RFQof the ion products received from the first RFQ.

DETAILED DESCRIPTION OF THE INVENTION

As used herein, “a” or “an” means one or more. Unless otherwiseindicated, the singular contains the plural and the plural contains thesingular.

As used herein, an “Radio Frequency (RF) Ion Guide” is a distinct groupor cluster of several electrodes (rods) located around straight channelof ion transport with RF potential differences applied between adjacentpairs of these rods. In case the number of these rods is four RF-ionguide is called Radio-Frequency Quadrupole (RFQ) ion guide. RFM refersto Radio-Frequency Multipole one example of which contains eight rodsand is called a RF-octapole.

RFM-ion guides as used in this application are built by segmenting eachrod of the RFM into discrete electrode sections so that different DCvoltages, and/or AC (RF) voltages can be applied separately or in anycombination to cool, trap, and/or rotate ions along or around the gasfilled longitudinal axis of the RFM, and providing of the desiredcomposition of supersonic gas flow along it axis may be used fortrapping of some types of the ions, isolating of the desired ions fromthe trapped ones, their transformation and delivering of the products ofthese transformations to downstream instruments and devices.

As used herein, IM or a “mobility cell” or “ion mobility cell” isdefined as a single or multi-channel device which performs mobilityseparation of ions.

As used herein “exit tube” or “exit capillary” is a segmented section ofelectrodes each of which electrode segment is individually electricallybiased and each of which electrode segment has a diameter less than apreceding electrode diameter within the IM cell to which it is fluidlycoupled, said “exit tube” or “exit capillary” being used allowdifferential pumping of the carrier gas exiting the IM cell before thisgas can enter a TOFMS and where said exit tube in some cases is arrangedto produce an adiabatic supersonic gas flow.

As used herein, “MALDI” means matrix assisted laser desorptionionization.

As used herein, “SIMS” means secondary ion mass spectrometry.

As used herein, “FAB” means fast atom bombardment mass spectrometry.

As used herein, the term “TOFMS” is defined as a time-of-flight massspectrometer both linear or reflectron type; as used herein, “oTOFMS” isdefined as a time-of-flight mass spectrometer both linear or reflectrontype configured orthogonally to the analytical axis of a precedinginstrumental platform such as, for example, the separation axis of anion mobility cell; “LoTOFMS” is defined as linear oTOFMS.

As used herein IM-oTOFMS refers to a combination of an Ion mobilityspectrometer with an orthogonal time of flight mass spectrometer.

The present invention relates generally to instrumentation andmethodology for characterization of chemical samples based on ionmobility spectrometry (IMS) and mass spectrometry (MS). Specifically,the invention relates to improved IMS, using the concepts of a new typeof interfacing of ion mobility cells with an orthogonal injectiontime-of-flight mass spectrometer (TOFMS) based on significant interfacecooling of the ion beam by combining an adiabatic supersonic gas flowwith a focusing radio-frequency quadrupole (RFQ) or multipole (RFM) ionguide. The improvements also include the capability to initiate chemicaltransformations within separated or isolated ions under high or lowtemperature conditions via their collisions with atoms, molecules andclusters which are either present or have been intentionally introducedand/or which were intentionally formed within the adiabatic gas flow.Structural and conformational information about the separated isolatedions can be obtained over a wide temperature region ranging from closeto absolute zero up to the limit of the thermal stability of theisolated ions. Measurement of a variety of physical characteristicsincluding primary and secondary structures (as well as the ratio of ionvolume to charge) is now possible over this wide temperature range.Moreover, a new type of separation of biomolecules in the gas phase isdescribed based on creating conditions in the neutral-ion reactor nearthe isoelectric point for the molecule of interest. Such possibilitiesare possible because of a new design of an ion mobility cell/TOFMSinterface comprising a well collimated cooled gas flow, a means ofcontrolling the gas phase composition and a unique RF-ion guide whichprovides controlled retarding and accelerating DC electric fields aswell as an AC rotating field. An electron impact ionizer located afterthe RF-ion guide allows measurement and control of the composition andproperties of the gas flow allows additional structural informationabout the ions and their products which are being manipulated inside theRF-ion guide. Additionally, the technique of multi-channel datarecording provides kinetic and structural information. The presentinvention enables methods both for increasing the dynamic range of themeasurements and for obtaining additional ion shape analysis beyond thatavailable from ion mobility alone. These improvements may be used toincrease the information content and the throughput of ions from asample into downstream instruments. The resulting instruments andmethods are useful for qualitative and/or quantitative chemical andbiological analysis.

A new approach for isolation and subsequent rotation of ions in a gasfilled RFM, is at the heart of this invention and is now described. Itshould be understood that while an RFQ is described for illustrativepurposes, other RFMs (such as RF octopole, etc.) may be used. Analyteions can be entrained in a narrowly divergent divergent gas beam created(for example) in a capillary interface between an ion mobility cell anda mass spectrometer said gas beam being formed during the expansion ofthe mobility carrier gas from high pressure to low pressure. If at thepoint just after the gas expansion (where the gas beam is narrowest) theentrained ions are then pulled from this gas beam by a momentaryelectric or magnetic force into a quadrupolar (or multipole) region,then these ejected ions may be made to rotate in a gas density which isalmost that of the residual gas in the interface and is several ordersof magnitude less than the pressure within the cooled beam from whichthe ions originated. Thus, ion rotation may take place in a spatialregion of almost constant residual gas pressure outside of and aroundthe dense gas beam which is diverging along and around the central(longitudinal) axis of the RFM. It is thus possible to trap and purifyparent ions and, if desired, further manipulate these purified ions(fragmentation, gas phase ion/molecule reaction, electron attachment)followed by subsequent reinsertion of the purified parent ions or theirreaction products into the neutral gas beam for further manipulation andtransport either to an MS or to other traps or into additional IM cellsor combinations thereof. There are some substantial advantages toidentifying and manipulating ions (particularly IM separated ions) byrotating the ions in a constant residual gas pressure when compared totraditional RF traps and cooling devices wherein ions are made tooscillate in and out of the gas beam (as in the conventional motionwithin a quadrupole). The stable orbit of an ion in a gas-filledrotating field region is determined both by its ion mobilitycrosssection and its mass/charge ratio. Thus, the coupling of such arotating field ion trap is particularly useful at the end of a mobilitycell which injects ions into the rotating region which have beenpre-selected by their IM cross-section. Simple theoreticalconsiderations, confirmed by computer simulations and experimentalmeasurements, allow the following main expressions which describe theproperties of ion rotation within a constant gas density.

The resonant rotation angular frequency {circumflex over (ω)}_(rot) forgiven ion charge ze (e is an elementary charge), mass m, RF-voltageamplitude V_(rf), internal radius of RFQ r₀ and angular frequency ofRF-voltage ω for small enough gas density (valid for the cases underconsideration) is given by:

$\begin{matrix}{{\hat{\omega}}_{rot} = {\sqrt{2}{\frac{{zeV}_{rf}}{{mr}_{0}^{2}\omega}.}}} & (1)\end{matrix}$

The resonant rotating radius for a given m/z value is proportional tothe mobility value K for given ion and amplitude of rotating voltageV_(rot):

$r_{res} = {\frac{{Kmr}_{0}V_{rot}\omega}{{zeV}_{{rf}\;}\sqrt{2}}.}$

The value

$\frac{Km}{ze} = \tau_{v}$

is a relaxation velocity time for the given ion at a particular gasdensity as described in the following section. 2τ_(ν) is acharacteristic time for achieving a steady state rotation of ions. Theradii of rotation for other ions with different m/z values (and the samemobility) should follow a bell shaped peak (close to Lorentzian incharacter) with relative width at half height given by:

${\frac{\Delta \left( {m/z} \right)}{m/z} = \frac{2\sqrt{2}}{\tau_{v}q_{M}\omega}},$

where the Mathieu parameter

$q_{M} = {4\frac{{zeV}_{rf}}{{mr}_{0}^{2}\omega^{2}}}$

should not be more than 0.7 for stable motion of a given ion. Thus themaximum possible resolving power for rotational excitation

$R_{rot} = \frac{m/z}{\Delta \left( {m/z} \right)}$

is given by:

R_(rot)≦0.247τ_(ν)ω.

The relaxation velocity time may be estimated through average collisionfrequency ν and consequently through collision cross section σ, gasdensity n, and average thermal velocity V of the carrier gas atoms andtheir mass M:

${\tau_{v} \approx \frac{m}{Mv}} = {\frac{m}{{Mn}\; \sigma \; \overset{\_}{V}}.}$

An example calculation of R_(rot) can be estimated for the followingexperimental conditions: For an analyte mass m=1000 Da, carrier gas HeM=4 Da, n=3.5×10¹³ for 1 mTorr residual He gas pressure, σ=3×10⁻¹⁴ cm²(crosssection of analyte in He), and an average velocity of helium atomsfor room temperature V=126000 cm/sec we obtain τ_(ν)≈1.9 msec, and forthe angular frequency ω=2τ·2 MHz R_(rot)≦5800. For larger frequenciesand also larger RF voltages (or less gas density) the resolving powerwould be even greater. The resolving power for larger ions should alsoincrease as a function of m/z since the mass of the ions is expected togrow faster than their collision cross section. Thus, the method of ionrotation has significant advantages for structural determinations of IMseparated ions since other approaches to ion purification and excitationmove the analyte ions for some time near the dense gas flow axis thusseverely limiting their resolving power. However, there are factorswhich limit the resolving power of the rotational method. Equation (1)which gives the resonant rotating frequency shows that a variation in r₀² (the squared internal RFM radius) may control the resolving power ifthe manufacturing precision of the RFM is not good. Also, additionalvoltages, if improperly applied to some RFQ sections for the retardationor acceleration of ions out of the gas stream and for trapping of theions may also result in some losses of rotational resolving power. Thereare two important rules to follow when applying such voltages. The firstone is that a uniform electric field along the RFM satisfies the Laplaceequation by having a zero component in the orthogonal plane. Thus thisfield will not have an influence on the ion rotation. The secondfavorable situation is a linear increase or decrease of the electricfield along the RFQ axis. Such fields would change the rotatingfrequency of the ions but would not change the character of harmonicmotion of ions (on average). Thus in such an ideal case the resolvingpower would not change noticeably especially if this field is not verystrong. Therefore the use of such fields for trapping rotating ions isquite feasible. It is interesting to note that AC harmonic fields with alinearly changing amplitude directed along RFQ axis also do not change(on average) the harmonic character of ion motion. The reason for thatis the known quadratic dependence of the effective potential of aharmonic AC field on its amplitude (this property was described for thefirst time (to our knowledge) in the book “Mechanika” by Landau andLifshitz published in Russia in the mid 1950's in which Petr Kapitsa,described the idea of such influences of fast field oscillations. In ourcase the amplitude can be changed linearly. After estimating theeffective force by first calculating the gradient a linearly changingforce would be present along the axis and in the orthogonal plane too.The only difference when compared to a DC field alone is that the forcein the orthogonal plane is always a focusing force. The main restrictionfor the possible number of rotating ions of given m/z value and mobilityis the space charge influence. As rotating ions with different m/zand/or mobility values are located at each time in different locationsinside the RFM their mutual space charge influence simple calculationssuggest that the accumulation of 1 000 000 or more rotating singlecharged ions over wide range of m/z and mobility values may be possiblewithin a linear RFM of 10 cm length. In analogy to the situation for theusual application of RF ion traps, the combination of several harmonicrotating fields along the linear RFM should independently excite ionmotion at different locations along the RFM.

The present invention deals with systems and methods using ion mobilitydrift cells for transporting ions through a high pressure gas to aTOFMS. The following concepts are described in various embodiments ofthe present invention: (a) controllable production of chemicaltransformations of separated or isolated ions under high or lowtemperature conditions via their collisions with atoms, molecules andclusters included or formed inside adiabatic gas flow, (b) extractinginformation about primary, secondary structures and the spatial shape ofthe ions over a wide range of temperatures from the close to absolutezero up to the temperature defined by the limit of thermal stability ofthe ions, (c) multi-channel data recording which is essential forobtained kinetic data processing and which allows also to increase theefficiency of sample use by obtaining as much useful information aspossible about the sample in a reasonably short time, and (d) a new typeof separation of biomolecules in gas phase based on providing conditionsin neutral-ion reactor close to the isoelectric point for the moleculeof interest. Specifically, the improvements lie in (i) use of a newdesign mobility cell/TOFMS interface comprising well-collimatedsupersonic gas flow and an original RF-ion guide which has thecapability of creating controlled DC and additional RF electric fieldscombined with an AC rotating field (ii) controlled variation of thebuffer gas composition inside the RF-ion guide by providing controlledamounts of larger reagent gas admixtures to the main ion mobilitycarrier gas (carrier gas is usually helium for the most ofapplications), (iii) accelerating, retarding or trapping of targetion(s) by appropriate DC and/or RF axial fields and AC rotating fieldinside RF-ion guide, (iv) removing undesired trapped or moving ions byresonant rotating field, (v) high selective isolation of ions with adesired m/z and mobility values by joint influence of resonant rotatingfield, DC fields and supersonic gas flow, (vi) controlled heating ofions by intentionally adding admixtures of relatively heavy atoms to thecarrier gas flow (Ar for example when added into a He flow will have tentimes more energy than He atoms), (vii) single collision induceddissociation of desired ions by addition of a controlled amount ofsignificantly larger atoms such as Xe to the carrier gas flow, (viii)performing different chemical transformations of ions for a giventemperature and time such as cluster formation, H/D exchange, ionmolecule reaction, noncovalent complex formation by addition to thecarrier gas flow of corresponding reagents, (ix) use of electronbombardment ionization of the combined gas flows for estimation of thegas flow parameters, for purity control of gases and for compositioncontrol of their mixtures as well as for ionization of neutrals (whichare usually not measured as reaction products during the iontransformations), (x) by changing the kinetic energy of electrons it ispossible either to produce electron attachment dissociation of ionsand/or to record ion appearance curves as a function of electron kineticenergy for dissociation of different bonds in the ions which may giveestimations for local electric field in the ion by comparison withkinetic data for thermal decomposition of the same bonds (xi) directmeasurements of isolated ion collision cross section over a controlledtemperature range from near absolute zero up to high temperatures (closeto the dissociation temperature for the ion), (xii) adjusting thecomposition of the gas flow to provide conditions in the neutral-ionreactor close to the isoelectric point for the molecule or species ofinterest and performing on that basis a new type of gas phase separationof biomolecules, and (xiii) use of a unique bipolar time-of-flight massspectrometer which is able to simultaneous record both positive andnegative ions and coincidences between them. Steps (iii)-(viii) may berepeated the desired number of times to isolate and manipulate (e.g.fragment) selected ions. Thus the method includes the new possibility ofMS^(n) of primary and product ions which have been selected at each stepboth on the basis of ion mobility cross-section and m/z (with moderateto high resolving power).

In comparison to conventional methods of temperature programmedcollision induced dissociation, the improvement described in (vi) aboveprovides a significantly more precise yet simple procedure forestablishing the desired temperature of the selected ions which in turnimproves the accuracy of kinetic measurements. Likewise, the new methodof ion decomposition described in (vii) above is more effective fordissociation of desired bonds in the ions as it relies on randomlydistributed single collisions to heat ions to a chosen temperature. Thisprocedure selectively cleaves the weaker chemical bonds in the chosenion. Product ions with a smaller ratio of charge to collision crosssection than the isolated parent ions would be relatively immune fromdecomposition. Since the smaller product ions would be able to overcomethe potential barrier retarding the isolated ions they would exit thespatial regions where the parent ions are being heated and decomposedsince their velocity would gradually increase to the velocity of the gasflow. If a further investigation of these product ions is desired (suchas subsequent decomposition for MS²) they can be isolated from theheating procedure before they are decomposed and stored for further useby putting a stronger retarding potential at some distance from thefirst retarding field which was (used to isolate the parents) so that a“shifting” field is created between the corresponding opposite sectionsof RFQ-RFM rods. This shifting field removes and conserves the product(daughter) ions during the time necessary to complete the decompositionor transformation of the desired isolated parent ions before thedaughters ions are further decompose in the heated region of the gasflow. Likewise, ion products with larger values of charge-to-collisioncross section ratio than the parent would move from the position wherethey were created as the trapped parent ions is being decomposed orreacted with a neutral gas to a position where the force of the electricfield on these product ions is compensated by the gas flow. Therefore,for product ions of larger cross-section than the parent a second“shifting” field can be created in the opposite direction which wouldalso remove these ions from the flow axis so that they too would not beheated and decomposed further. These two opposite fields located at bothsides of the region in which the parent ions are isolated will notsignificantly change the location of the parent ions so their heatingand transformation (e.g. fragmentation) may be continued. Some of thevery small product ions may come to the rods and be lost, but usuallysufficient structural information about large parent ion species such asbiopolymers may be obtained from product ions not much less than half ofthe m/z of the parent ions. Improvements (viii) and (xi) provide aunique method for the measurements over an unprecedented temperatureinterval—from the region close to absolute zero to the values up to andexceeding where ion decomposition can occur—which is difficult orimpossible to do by other methods. Furthermore, trapping ions of a givenmobility and/or a given m/z value can be accomplished with considerablyreduced restriction from space-charge compared to existing conventionalmethods since ions are trapped only for a narrow interval of mobilityand/or m/z values. This feature has implications not only for analysis,but perhaps as importantly for preparatory scale purification andselected area deposition of mobility and m/z selected molecules.Moreover, isolation and storage of a variety of desired ions by this newmethod does not necessitate the loss of other larger or smaller ions asis mandatory with other trapping methods. We pick certain desired ionsout of the gas stream while retaining the others within the gas streamwhich forces their transport either to a mass spectrometer for analysisor to other RFM traps in series for further refinement.

There are at least four possible methods of isolation of desired ionswhich may be chosen given the demands of specific experimentalsituations. 1) The simplest method removes undesired ions from thetrapped desired ions with a resonant rotating field determined for aspecific chosen interval of ion mobility. This method demands only theknowledge of m/z of trapped ions and gaseous conditions in the region oftrapping. Here undesired ions are removed from the trapped ones and thusthis method is similar in result to other methods of isolation ofdesired ions within a trap. The time for ion removing for some set ofm/z values may be large enough or demand using of more complicated powersupply for providing sums of rotating fields with given set offrequencies instead of device for producing of a single harmonicrotating field. 2) Alternatively, it is possible to excite the rotationof desired ions in a way which shifts them into a stable orbit within aregion of reduced residual gas pressure around the main gas flow. Thisallows the undesired trapped ions which remain behind in the gas flow tobe transported by this gas flow to a serial RFM or directly into a massspectrometer by reduction of the retarding trapping potential. Once thedesired ions flow away, subsequently reapplying the retarding trappingvoltage while simultaneously stopping the rotating field willre-introduce the desired ions back into their trapped position withinthe gas stream where they would be ready for further manipulation ortransformations. Here no ions are lost for measurements. To implementthis method it is necessary to know the pressure gradients within theRFM, the m/z value, and the mobility coefficient (collision crosssection) of these desired ions. The ion mobility is already give by theretention time within the ion mobility separation which is used. Bothapproaches (1 and 2) may be time consuming and inconvenient for classicmobility separation of ions when mobility peaks are relatively short intime and resulting mobility/mass spectra are accumulated by summingmobility separated ions over a repeated number of introduction of sampleions into the ion-mobility mass spectrometer. Thus, it may sometimes beundesirable to repeatedly perform the procedures of ion trapping,isolation and transformation after each and every each sample ionintroduction. 3) For continuously trapping specific desired (target)ions from the sample of ions which are being repetitively introducedinto the IM-TOFMS, it is possible to continuously maintain the resonantrotating field throughout the acquisition time of the experiment. Inthis case only ions with chosen m/z value would come out of the mainstream of the gas flow and would move along RFQ-RFM ion guide veryslowly and independently of the other ions remaining in the gas flow.Providing a small accelerating field at the beginning of the guidefollowed at some distance along the RFQ-RFM axis by a retardingpotential which is weak enough not stop any ions within the on-axisdense gas flow while continuously applying an appropriate rotationalfield around the RFQ axis in the region between these two fields willlock the desired ions in a rotational orbit outside the gas stream andbetween the two fields. 4) Alternatively it is possible to trap ionswithin a resonant rotational excitation as the ions with desiredmobility are entering the RF-ion guide. A retarding potential should beapplied at all times except when the desired ions move into thebeginning of the ion guide. This retarding potential will prevent ionsfrom drifting outside the main gas stream and force their motion insidethe stream at the beginning of the ion guide. Just as the desired ionsenter the RFQ the polarity of the retarding potential changes is reversewhich moves desired ions out of the flow into a region influenced by aweak rotating field which is effective only in the residual gas pressureoutside the main stream gas flow. After the time for extracting of thedesired ions has passed, the rotating ions are shifted by application ofpotential gradients from the beginning of the ion guide and trappedbetween two opposed retarding potentials further along the RFQ axis.

Finally, a new type of ion separation and isolation, not normallypossible with mass spectrometry, results when conditions within the gasflow are created close to the isoelectric point for the selectedmolecules in the gas flow. This approach is described in detail herein.Furthermore this type of biomolecule separation will simultaneouslypresent both positive and negative ions to the mass spectrometer;therefore, a new scheme for bipolar time-of-flight mass spectrometerwhich is able to simultaneous record both positive and negative ions(and coincidences between these positive and negative ions) is essentialfor gaining maximum information from the new separations. This new massspectrometer and its coupling to the newly enabled separation methods isalso described herein.

These improvements may be used to increase throughput from an ion sourceto downstream instruments and they may also provide additionalinformation about the samples. The resulting instruments and methods areuseful for qualitative and/or quantitative chemical and biologicalanalysis.

In the present invention ions of interest are isolated using both theirmobility and m/z values. Isolation of the desired molecules on the basisof their ability to form different ionic forms under controlledconditions in the gas phase which, in conjunction with significantlyimproved control of the experimental reaction parameters determining thespecific ion transformations, gives an opportunity for measuring uniquestructural information about ions present in the original samples.

When ion mobility cells, filled with a few Torr of buffer gas, are usedas a volume/charge separation stage in front of a mass spectrometer, thecooled ions exit through a small exit aperture or exit capillary into adifferentially pumped low pressure region before entering the highvacuum region of the mass spectrometer. To minimize transmission lossesthrough the small aperture, the ion beam inside the mobility cell mustbe focused. Ion beams should be as narrow as possible in the regionbetween the mobility cell and TOFMS to allow the use of smalldifferential pumping apertures (enabling lower gas flow) and to achievehigher mass resolution for TOFMS operation. Therefore the beam should becooled as much as possible to obtain low divergence. As described inco-pending U.S. patent application Ser. No. 11/441,766 filed May 26,2006, (Multi-Beam Ion Mobility Time-of-Flight Mass Spectrometry withMulti-Channel Data Recording) cooling can be done by directing a gasflow containing mobility separated ions through a narrow exit tube (ortubes for multi-beam systems) which separate the high pressure ionmobility region from the differential pumping region prior to the massspectrometer. By such a procedure, the gas and ion stream case canemerge with an angular divergence corresponding to a temperature of 1Kor less and the ion beam traveling in such an adiabatic supersonic gasflow can be further focused to even smaller final diameters by the useof an RF-ion guide as suggested in U.S. patent application Ser. No.11/441,766, filed May 26, 2006.

FIG. 1 shows the improvements of this interface region over the priorart by making an the RF-ion guide wherein the rods of the RFM containmultiple electrically isolated sections so that it is possible to createany combination of variable DC and AC fields inside the RF-ion guide.These fields may be arranged as will be described herein in order fortrapping and isolation of selected ions and for transforming theseisolated trapped ions by collisions with some admixture gases added tothe buffer gas flow. This approach of trapping and transforming may beused in structural investigations of samples of different nature. Thetransformation of the trapped ions may include fragmentation as aspecial case, but will also include a variety of different ion-moleculeand or ion-ion gas phase reactions. Two properties of the RF-ion guideshown in FIG. 1 should be noted: A) there exists a large difference ingas density exists along the center axis of the RFM within the directedgas flow compared to the residual gas density in the region radiallyoutward from the RFQ axis; therefore, small electric fields can have astrong influence on ions outside of the main gas stream yet will notsignificantly disturb the motion of ions which remain inside the gasflow along the RFM axis, and B) small admixtures of heavier atoms (5,6)and molecules intentionally combined with the main carrier gas flow(usually helium (8)) will not significantly change the gas flow velocityand the temperature of this carrier gas (8). After some distance alongthe RFM axis after the last electrode (61) of the exit capillary, theseheavier admixtures (5) will have attained the same drift velocity as thelighter buffer gas flow (8). Therefore, this means that heavy admixturegases have less divergence (by a factor equal to the square root of theratio of their masses) than the buffer gas (e.g. helium buffer gas).Thus, the heavy gas density relative to the density of the lighterbuffer gas would increase as their distance from the exit tubeincreased. The density of heavy admixture atoms (or molecules) along theaxis of the flow approaches a limit which is equal to the product ofinitial admixture relative density and the ratio of its atomic (ormolecular) mass to the mass of the main gas atoms. For example for Ar(5) admixture atoms in He (8) this factor of increasing relative densityalong the axis of the flow would be 10. During and after the expansionthe admixture (atoms) rapidly acquire the average velocity of thepredominate helium buffer gas atoms; therefore, the admixture atomsenergy of motion is more than that of helium atoms by the ratio of theirmasses. Thus, if an ion (12) exiting from the IM cell (or any other ionsource) into the RF guide is intentionally stopped or retarded near theflow axis (in a region of high relative admixture density) within thiscombined He (8) buffer and argon (5) admixture gas flow then thisretarded ion (12) may be effectively heated by collisions with theseheavier admixture atoms (5); furthermore, the temperature of thisheating is not expected to be as dependent on the chemical andstructural characteristics of the ions when compared to a moretraditional method of ion heating which occurs, for example, in CID(collision induced dissociation). In traditionally applied CID, ions aredecomposed by moving them within a gas under the influence of a variableelectric field and variable gas density CID entails a wide variety ofcenter of mass collision energies between the ion and the inert gas).Beginning at a distance along the flow axis (which is far enough fromthe tube exit to ensure that the admixture atoms and the entrained ionshave come to equilibrium velocity with the He) the temperature of thision heating will depend with good accuracy only on the relative velocityof ions and the gas flow. Shifting of ions and their dissociationproduct ions away from the gas flow axis would reduce this temperatureonly slightly proportional to the damping of a narrow Gaussian densitydistribution of the larger atoms or molecules.

In FIG. 1 an IM cell (2) is fluidly coupled to a gas dynamic interface(7) by an exit capillary (3) comprising gas tight individuallyelectrically isolated and biasable electrodes (61). The cross sectionshown in (FIG. 1) of the gas dynamic interface (7) comprises an RFQ(300) (two rods of which are shown schematically in cross section eachconstructed by 16 independently biaseable electrode sections (30)). Theinterface (7) provides for differential pumping (15) and for processingand forming an ion beam (19) for ultimate injection into a detector.This detector can be a oTOFMS with multi-channel data recording which isused for different embodiments for analysis of ions of both signsdirectly produced by ion sources or after post-ionization of neutralswithin the trapping region as is described, for example, in co-pendingU.S. patent application Ser. No. 11/441,766, filed May 26, 2006 entitled“Multi-Beam Ion Mobility Time-of-Flight Mass Spectrometry withMulti-Channel Data Recording” and U.S. patent application Ser. No.11/441,768 filed May 26, 2006 entitled “Multi-Beam Ion MobilityTime-of-Flight Mass Spectrometer with Bipolar Ion Extraction andZwitterion Detection”. Ions entrained within a gas flow (1) (normallyhelium) are directed from the IM cell (2) into a sectioned exitcapillary (3) where adiabatic supersonic gas flow is formed and ionsmove under the combined influence of accelerating gas flow and electricfields along the tube (3) provided by biasing to the individualcapillary electrode elements (61) ( five electrodes (61) are illustratedin FIG. 1). Preferably, the surfaces of the electrode sections insidethis tube should be coated with thin dielectric films and charged beforethe experiment by charges of the same sign to reflect ions from thewalls or may be fabricated from a piezoelectric device which can bebiased to transmit ions of both signs. This capillary exit (3) hasseveral valved (4) input orifices for inserting different gaseousadmixtures (5,6) into the gas flow. The delivery valves (4) undercomputer control should provide the desired flows of these admixtures.As examples of these admixtures atoms of Ar (5) and Xe (6) are shownsymbolically. The delivery valve for Ar (5) is shown open and othervalves are closed. Obviously more than two valves each with its ownspecial gas may be incorporated in this section. After the exit tube thesupersonic gas flow containing both entrained ions and intentionaladmixtures is inserted into sectioned RFQ ion guide (300) equipped withsectioned multielectrode rods (30), and is pumped by differentialpumping (15). In the ion guide (7), mobility separated ions (10), (12)and (13) are focused to the axis of the flow under influence ofRF-voltage and gradually acquire the drift velocity and the temperatureclose to those of the gas flow. As mobility separated ions of interestenter the gas flow, different voltages (DC, AC and RF) can beindividually or collectively applied to each of the electricallyisolated sections of the RF-quadrupole rods (31). DC retarding voltagesare intermittently applied at chosen times to electrode sections (31,37)of the bottom and top rods in the middle of the RFQ (300) so that aretarding field (14) is created which stops the ions with a mediummobility (12). The “small” ions (10) will have already passed thisregion and are not affected by this field while “large” ions (13) areable to overcome this retarding field due to the higher energy theyobtain in the adiabatic expansion relative to the the medium sized ions(12) which are made stationary by the field. These stationary ions (12)may then be heated to a desired temperature by seeding argon atoms (5)into the carrier gas (e.g. He) gas flow (8) with a velocity close to1400 m/sec (average quadratic velocity of helium for the roomtemperature ˜300 K). This corresponds to 3000 K for kinetic energy ofthe Ar atoms. Thus 10% admixture of Ar passing through and collidingwith the stationary ions (12) would give about 600 K of ion temperature.By variation of the Ar abundance in He, the desired temperature of thestationary ions may be achieved. To measure and control the ratio ofAr/He in the gas flow an electron impact ionizer with electron emissivecathode (20) and anode (21) is located just after exit orifice (18) ofgas dynamic interface (7). The gas and ion flow (19) which is enteringthe oTOFMS is ionized by rectangular pulses of electrons created byapplying a voltage (22) to the anode (21). The He⁺ buffer gas and Ar⁺admixture ion intensity created by the electron impact ionization can bemeasured with the oTOFMS in the same process as IM separated ions (19)are being recorded so that the rare gas ion signals can be used tocalibrated the concentrations of He and Ar gas in the gas beam which wasused to process an manipulate the IM separated ions (19). A small areaorifice (18) and adequate pumping (23) provides a low operationalpressure inside the oTOFMS.

Another important phenomenon to be considered in a gas dynamic interfaceis the capability for formation of ion clusters (11) with admixturemolecules at the end of the RFQ (300). To investigate formation andfragmentation of these clusters (11) (which may give additionalinformation about ion conformation at low temperatures) electric fields(24) and (16) may be varied giving various intensity distributions ofcluster ions. For the most accurate quantitative information aboutcluster formation, the resulting electric field distribution along gasflow axis should be proportional to the gas density to provide constantion drift velocity and consequently constant temperature of the ions.This approach is described in more detail much later in thisapplication. To increase transmission of these ions through exit orifice(18) additional short range electric field may be created (17).

FIG. 2 is a cross section taken through electrodes 31 and 37 of FIG. 1and shows how a rotating RF field can be applied in addition to the DCvoltage and RF cooling voltage already applied on these electrodes. Inone embodiment of the present invention, a resonant rotating field isused for removing undesired ions (32) from the trapped ions (12) asshown in FIG. 2. This field is created by applying an AC harmonicvoltage with phase shift π/2 between adjacent rod segments (31), (34),(37), (36), within the RFQ (300). Our previous experience in usingrotating fields for heating and decomposing ions (Raznikov, et. al.,RCM, 15, 1912-1921, 2001) shows that a mass resolving power of a fewhundred (FWHM) may be achieved for such a case as shown in FIG. 2 wherethe trapped ions remain in the dense gas flow. Further purification ofthe trapped ions (12) may be achieved by re-exciting ion rotation withsome rotating frequency appropriate for the m/z of any undesired ionsremaining in the trapping region.

Alternatively it is possible all at once to generate the rotating fieldas a sum of resonant rotating fields for ions with all undesired m/zvalues. It is possible to excite resonant rotation of just the desiredions from all the trapped ones instead of removing all ions but thedesired. Thus the desired ion comes out of the gas flow and theundesired remain trapped. The amplitude of the rotating field in thiscase should provide enough displacement of the desired ions from theaxis of the gas flow to maneuver them into a region out of the dense gasflow and into a region where the gas density is noticeably less than onthe axis. This RF amplitude must be chosen carefully because if toolarge then the desired ions will rotate into the rods where they wouldbe lost. If the RF amplitude is appropriate, ions will start to driftbackward as they transition out of the main gas flow under the influenceof retarding field and finally they would stop their drift as they arecompletely moved out of the gas flow and they would then rotate in astable orbit around the gas flow. In the case of a weak retarding fieldwhich decreases slowly in the direction opposite to the gas flow, thelocation of the desired rotating ions may be removed upstream far enoughfrom the trapping region (where the gas beam diameter is minimum) sothat only undesired trapped ions are released when the retardingtrapping potential is switched off for a time just sufficient forreleasing the undesired ions but not the desired rotating ions.Switching off the rotating field after restoration of the retardingfield would result in re-isolation of the purified desired ions back inthe gas flow and in the trapping region where further manipulation andtransformation of the purified ions could occur.

After isolation of the desired ions (43) in a trapping region by theretarding fields (14) which are shown in FIG. 3 the ions may then besubjected to different transformations. For example, collision induceddissociation may be provoked by a single impact of relatively highenergy large atoms (Xe)-(6). Ions (43) are heated by Ar (5) collisionsto high enough temperature to be almost ready for the decomposition. Ifthere is no desire to further isolate individual product ions foradditional investigations then only RF focusing voltages are applied tothe rods (7) of the RFQ. The “small” dissociation products (44) (with acollision cross section to charge ratio less then that for parent ions)will be removed from the flow axis under influence of the field (49)between opposite RFQ sections of rods; therefore, they would not besubjected to further decompositions by heavy admixture atoms which havesignificantly less divergence than that of the helium atoms. “Large”product ions (with a collision cross section to charge ratio more thenthat for the isolated parent ion) would overcome the retarding field(14). Under the influence of stronger retarding field (41) and the field(50) being opposite to the field (49) but created between the othersections of RFQ rods they (42) would be removed from the axis too. Thus,with the “large” and “small” product ions being continuously removed,the main dissociation mechanism is by single heavy atom (Xe) collisionswith individual parent ions (which have been preheated near dissocationby previous Ar collisions). To isolate the ions with desired m/z valuefrom the decomposition products a graded rotating resonant field shouldbe created between RFQ rods. The amplitude of this U_(rot) field shouldbe not uniform along RFQ because the gas density along the axis of theRFZ is not uniform. The variation of the amplitude of this rotatingfield along the RFQ axis may be found empirically and may be close tothat shown in the plot (47). This type of rotating field distribution isessential also for isolating of desired ions from the trapped ones shownin FIG. 1. To remove ions from the region close to the center of theflow a strong enough field is necessary. After coming to the regionswith less gas flow and density the ions would be shifted to the frontend of the RFQ by a decreasing electric field (48) and here lessrotating field is required for stable ion rotation. Linearly decreasingthe DC field (48) is preferred as it creates a linear increasingorthogonal field directed from the axis of RFQ (this is a consequence ofthe Laplace equation for electric field potential). The resonantfrequency for rotation of ions with chosen m/z would be constant alongRFQ in this case as the influence of such field would be equivalent onlyto some decrease of RFQ effective potential and not change its relativedistribution. Ions with a given m/z value but with different collisioncross section (45) and (46) may ultimately rotate at different placesalong the axis inside the RFQ. Thus in favorable cases it may bepossible to distinguish between such product ions and even to decomposethem further and separately. By gradually removing DC potentialsdifferences between the left side and the middle of the RFQ as well asthe rotational RF until these potentials are set to zero then the ionswith larger collision cross section would be released first, return tothe gas flow and would be recorded by the TOFMS. If it is desired tochoose some of these remaining product ions for further transformationthen retarding DC potentials forming fields (14) and (41) should berestored just before the remaining desired ions begin to enter the gasflow. If there is no desire to work with the remaining rotating ions inthis cycle of measurements they may be removed by increasing theamplitude of the rotating field to move these ions to the rods to avoidconfusion with product ions of newly trapped primary ions.

FIG. 4 shows a cross section view of one unit of an RF multipole ionguide taken from an array of ion guides used in a Multibeam IM TOFMS(described in co-pending patent application U.S. Ser. No. 11/441,766filed May 26, 2006 and incorporated by reference herein) wherecylindrical rods (31) and (37) are separated by grounded plates (52). Tocreate DC and rotating fields independently for each ion beam, sectionedelectrode plates (55), (53), (54) and (55) isolated from ground (52) arealternated intersperse with the pairs of cylindrical rods (31,37). Alsoillustrated in FIG. 4 is the beginning of the isolation process justafter imposing a resonant rotating field onto some of the trappedproduct ions (45) and (46) (shown also in the previous FIG. 3) after thefirst fragmentation of the isolated parent ions (43) trapped in theregion of high gas density and exposed to the collisions with admixtureAr (5). Product ions (45), (46), (44), and (42) are shifted by a DC androtating fields from the region of heating where the parent ion (43) isshown. After releasing the product ions into the oTOFMS (where their m/zwill be measured) by switching out the retarding potential and therotating field, a new batch of isolated parent ions could be trapped andmade ready for further investigations. Alternatively desired fragmentions could be trapped and fragmented allowing MS² measurement to also beperformed in this case. Repetition of this procedure n times would givethe possibility of implementation of MS^(n) methods. This sequence ofrepeated trapping, fragmentation, and release and measurement of productions and repetition of this sequence is applicable to any of the RFMembodiments taught in this application.

FIG. 5 illustrates an alternative way of trapping and isolating thedesired ions by using resonant rotating fields and DC fields toselectively extract the a desired ion from multiple mobility separationsof ions. In the most universal form of IM spectrometry a source of ionsis pulsed and the pulsed ions drift through the mobility cell andseparate according to their densities (which is reflected in theirdiffering mobility drift times through the IM cell). In one example ofthis, a laser is used to pulse a MALDI ion source whose ion output isdirected into a IM cell. At some time after the initiating laser pulse,the mobility resolved ions begin to elute as a function of time afterthe initiating laser pulse. Application of a rotating field on the firstfew electrodes next to the first electrode (30) of the RFQ (300) whileintermittently applying a DC extraction between electrodes (61) and (30)will draw any ion which are eluting from the exit capillary (3). Thus bytiming the application of this DC field relative to the initiating MALDIlaser pulse, it is possible select ions with only a desired IM driftvelocity. For removing only these ions from the dense on axis gas streamion rotation is started ions at the beginning of the RFQ a field (65) iscreated between the final section (61) of the capillary tube and thefirst section of the RFQ (30). This field should be fairly weak so asnot to totally overcome the focusing effect of the RF-field and not tomove ions to RFQ rods. In this case ion rotation is initiated in theregion close to the end of the exit capillary (3) where the residual gasdensity is lowest and which should result in a significant increase ofmass resolving power with resonant ion rotation. For residual heliumpressure of about 1 mTorr this resolving power may be 4000 or more.Highly efficient dynamic trapping of ions, which simultaneously have adesired m/z value and some interval of mobility drift velocity, can beachieved by selecting a resonant rotating field with it frequency chosenfor selecting an m/z value and its amplitude chosen for a specificmobility cross-section which are then applied in conjunction with asmall retarding electric field (64) (created over some distance from thebeginning of the RFQ). The extraction field (65) is relaxed to zero andthe weak fields (64) are manipulated to shift the trapped desired ionsslightly downstream into a stable trapping region where they arepurified over time by the rotating RF field. Repetition of this sequenceof applying the timed extraction field (65) after the laser shot to movethe desired ions out of the dense gas stream, manipulating the desiredions (62) by using the weak field (64) to shift the desired ionsdownstream into a trapping region, followed by relaxing the extractionfield (65) to zero voltage to allow the next packet of desired ions toexit the capillary exit (3) allows filling of the trap with new (target)ions after each and every MALDI laser pulse. These new ions can beextracted and added to the already stored ions which can then all be andprocessed and stored for many seconds after the MALDI experiment isconcluded. The mobility cross-section interval which can be trapped in arotational orbit following this proceedure depends on the “radius” ofthe dense gas stream and the internal radius of the RFQ. For a realisticexample, the “radius” of the gas stream is assumed 0.5 mm near the exit(61) of the segmented exit capillary (3), and the average radius ofrotation of the desired ion is 1 mm inside the RFQ whose internal radiusis 2 mm. A rough approximation of the range of IM cross-sections whichcan be initially captured into the rotating filed trap at this locationis that ions with one half the mobility cross-section will expand intoan orbit which hits the rods and are lost and the ions with twice themobility cross-section will have an orbit which is too small to leavethe dense gas flow along the RFM axis. The retarding field (64) shouldth be chosen small enough not to prevent any ions to move inside the gasstream but strong enough to retain rotating ions outside the main streamof the gas flow. The field (65) at the beginning of RFQ would preventrotating ions from escaping from the end of the RFQ nearest the exitcapillary (61) or alternatively fields (64) can be manipulated to movethe rotating ions downstream where a trapping and storage region can beestablished. All of the other ions which have not been removed from thegas flow will consequently pass unimpeded under the rotating trappedions and through the exit aperture (18) and enter After finishing themeasurements of the ion MALDI-IM-oTOFMS spectra the laser ablation isterminated and the mass spectrometer is then used to process the unknowntrapped ions which have been selected and purified according to theirdesired m/z and mobility within the rotating trap region Trapped ionshaving the desired m/z may comprise several different types of ionshaving the same m/z and different mobility values. It possible toprocess this collection of trapped ions at this time while no other ionsare entering the buffer gas flow into the oTOFMS by using a programmedreduction of the rotating field amplitude to successively release ionsback into the gas flow according to their decreasing mobility crosssection. We first gradually decrease the rotating field amplitude to avalue at which we begin to record ions in the mass spectrometer and atthat point the rotating field amplitude is held constant until we nolonger see ions or alternatively as soon as we observe the first ions wenot only stop decreasing the rotating field amplitude but we alsoreinstate the retarding potential so that ions which are at this pointre-entering the gas stream are stopped in trapping region of the mainstream gas flow. After some delay time, which should be foundexperimentally, all the procedures for heating, transformation of ionsand recording of mass spectrum of the products are performed. After thatthe gradual decreasing of the rotation field resumes to release the ionswith higher mobility cross-section from their stable orbits and backinto the gas stream is resumed so that ions may re-enter the gas streamand be transmitted directly to the mass spectrometer (zero retardingfield) or further trapped and fragmented (maintaining RF rotating fieldamplitude constant and re-instating retarding field). When the next ionwith chosen m/z and is recorded all the procedures described above arerepeated once more. When the rotating field is zero the measurements arestopped at which point one has obtained the entire MALDI-IM-oTOFMSspectrum along with MS^(n) of the trapped ions all in one set of timingsequences followed by a timing period of releasing all the trapped ionsaccording to their collision cross-sections as well as any desired MS/MSspectra of the product ions. The procedures described around FIG. 5 mayresult in a reduction of time to perform the entire IM-MS^(n) experimentsince slow operations with gas flows may be repeated fewer number oftimes while still allowing the IM-MS measurements to proceed whiledesired ions are being accumulated. Manipulations on the trapped ionssuch as dissociation or reaction with neutral molecular adducts can thenproceeds after the MALDI-IM-oTOFMS spectrum is completed acquired.

Measurement of Mobility Cross-Section by Slow Release of RotationallyTrapped Ions Back Into the Gas Stream

FIG. 6 shows some modification to the previous approach with theadditional possibility to increase the resolution of cross-sectionmeasurement of the trapped ions using the RFM and rotational fieldtrapping. In order to avoid trapping ions with mobility coefficientsdifferent from the desired some retarding potential (66) is applied tothe first sections of RFQ rods all during the IM-oTOFMS acquisition timeperiod for recording the untrapped ions except during the brief time(few tens of microseconds) time when the ions with the desired mobilitycross-section are coming out of the exit tube (61). This potential (66)should be enough to prevent ions from moving outside the main stream ofthe gas flow but not so strong as to stop ion motion inside the mainstream. At the moment when the desired ions just begin to pass the firstsections of RFQ (7) the potentials on the first rods of RFQ are switchedto form the accelerating field (65) shown in FIG. 5. Thus the ions ofdesired mobility cross-section begin to be trapped by the resonantrotating field. After the last desired ions pass the starting plane ofRFQ (7) the potentials on RFQ sections are switched back to form theretarding fields (65) shown in FIG. 6.

After isolation of the ions by the method shown in FIG. 6 and in FIG. 5,In addition to recording the products of ion transformations, it ispossible also to get some information about collision cross sections ofthese ions over a wide under wide temperature range. This information isdifficult by standard mobility measurements. FIG. 7 illustrates onepossible way to obtain such estimations over the temperatures below roomtemperature down to the region close to absolute zero by forming adecreasing electric field (76) in the direction of the gas flow alongRFQ and no admixtures to He atoms (8) flow are added. For measuring attemperatures higher than room temperature some admixture of Ar (5) atomsmay be inserted into the He flow. The field decrease should correspondto the decreasing of the gas density along the flow so as to provideconstant drift velocity of ions above the velocity of the gas flow. Inconditions of low or moderate pressure mobility measurements this gasflow may be considered as a molecular beam coming from the exit tube(61) with the axial velocity close to the mean squared gas velocity forthe room temperature and some divergence corresponding to thetemperature of the gas. How to measure this axial velocity and theaverage angle of the gas divergence is explained below. Switching outthe rotating field makes previously trapped and rotating ions (62) tore-enter the gas flow as the increased in the retarding field (64) alsoprevents them to move along the flow and RF focusing field would pushthem to the axis. After some time sufficient to collect all ions nearthe axis the polarity of retarding field (64) is changed and itsstrength is adjusted to form at that place a slowly decreasing axialelectric field. Under the influence of the gas flow and the axialelectric field ions after some time would achieve the velocity above thevelocity of the gas flow. The value of this additional velocity for agiven field strength gives the mobility of the ions from which theircollision cross section may be estimated for known gas density. Thetemperature of the ions would be proportional to the square of thisadditional velocity and may be estimated from measurement to bedescribed in the following sections.

The value of the total ion velocity as well as the time of their motionafter switching the polarity of the retarding field (64) may beestimated via multichannel TOFMS data as also described below. Thevelocity of the gas flow and its density is measured by electron impactionization (20)-(21) of the carrier gas and any admixture atoms in themode shown in previous figures. This ionization is initiated by applyingan anode voltage pulse (77) (it is also possible to implement electroncapture dissociation technique for large multicharged ions in the casewhere the voltage pulse width is chosen just to “stop” electrons in theregion of the flow (19) coming out of the exit orifice (18)). A cathodewith indirect heating (75) gives a reduced energy spread of theelectrons and is desirable for use in this application. Sophisticatedand effective designs of a low energy electron ionizer are known in theart and may be found in the literature. This electron impact andelectron capture source enables estimation of the collision crosssection for the RFQ isolated ions in FIG. 7 for the same temperature bycreating the small decreasing retarding electric field of the samestrength which provides the same steady state absolute value of velocitydifference between ions and the gas flow. The comparison of the resultsof two such measurements will give the dynamics of the cross-sectionvariation as a function of ion temperature by measuring the change inthe relative velocity of ions and buffer gas atoms. Also it wouldprovide a way to estimate small changing of the ion velocity due tocollisions with gas after exit orifice (18). This effect is slightlydifferent for both cases since because the accelerated ions would movefaster after exit (18) they would then have fewer collisions in thisregion compared to the collisions of the retarded ions. Such an approachcombined with controlling the dense gas temperature by injecting thenecessary heavy admixture gas into the dense gas flow may also be usedfor investigation of the formation and desolvation of cluster ions underlow temperature conditions. Comparison of cluster ion distributionsmeasured for the same accelerating and retarding fields would enableestimation of “equilibrium” conditions for cluster formation.

The methods described above may be implemented not only in a single RFQion guide. It may be even more effective in some cases to use a combinedion guide such as, for example, those shown in FIG. 8 and in FIG. 9Using an RF-octapole (or multipole with number of poles more than 4) atthe beginning of ion guide for trapping, isolating and transformation ofions may be more preferable at least in some embodiments of the presentinvention. A faster production of an effective focusing potential foroctapole (ideally it is described by the polynomial of the fourth orderin contrast to second order polynomial for RFQ) would result in morenarrow distribution of rotating ions (82) and less probability of theirdischarge on octapole rods (81). Somewhat better separation of ions withlarger m/z is expected for RF-octapole. For creating rotational fieldsthe phase shifts between adjacent rods (81) of the octapole is π/4instead of π/2 used with the quadrupole. Product ions may easily producecorona like paths (84) under the influence of DC-fields directed to rods(81) since the effective focusing potential is reduced as a polynomialof the fourth of distance from the rods to the axis instead of thesecond order of this distance in the case of the RFQ. Thus these productions may faster come away from the region of heating by Ar atoms (5) anddecomposition by collisions with Xe atoms (6) thus providing evencleaner conditions for ion decomposition than when using the quadrupole.The final part of the ion guide may then be a quadrupole as it providesbetter focusing and cooling of the ions. Its rods (94) built of isolatedsections allow to also create an electric DC field inside to provide thepreviously mentioned functions of a low temperature reactor for primaryions and their products. As an example in FIG. 9, such product ions (93)may be an ion with less charge to cross section ratio than that for theprimary isolated ions (73). This product ion (93) can overcome theretarding electric field (14) and form some cluster ion (95) (eitherintrinsically or with intentionally added admixture gas) while movinginside the cooled gas flow. Under the influence of a de-clusterizationfield (96) this cluster ion may be decomposed partly (97) and kineticsof this decomposition may be investigated by changing of this field (96)and recording the resulting cluster ion distributions in the massspectrometer.

An important capability which is added to the oTOFMS allowing suchkinetic investigations is multichannel data recording and positionsensitive detection. The TOFMS may be reflectron type (see., e.g., U.S.Pat. No. 6,683,299 and U.S. Pat. No. 7,019,173, and published U.S.Patent Application 2005/0127289 A1, all of which are incorporated byreference as though fully set out herein) which is more suitable in ourcase or even a linear TOFMS. A schematic of a linear oTOFMS suitable forthe considered measurements is shown in FIG. 10. It is similar to thatdescribed in U.S. patent application Ser. No. 11/441,766 filed May 26,2006. Here for simplicity a single beam instrument is considered. Theimportant property of this instrument is the sequence of anodes (107)which is not shown finished in the figure. These anodes are connectedperiodically with corresponding channels of the TDC. For example for aneight channel TDC the first 8 anodes are connected sequentially to 8channels of TDC. The 9th anode is then connected in common to the 1stanode into the 1st channel of TDC, the 10th anode in common with the 2ndanode goes into the 2nd TDC channel and so on. The length of ion packageinserted into TOFMS is controlled to be less than the length defined bythe first eight anodes so no confusion of ion signals will occur.Alternatively, more TDC recording channels may be added so that eachanode is independently measured into its own recording channel.Furthermore, each anode may have its own TDC channel each channel ofwhich is capable both of measuring the arrival time and the analogintensity of the portion of the electron pulse from the MCP which landson one anode. When one ion strikes the ion detector, the resultingelectron cloud may be spread over multiple anodes so that, after chargecentroiding, the position and time of arrival of the ion at the detectorplane can be measured with high time and positional accuracy. Suchposition sensitive detectors are known in the art and their use allowseven more accuracy when making the angular and velocity measurementsdescribed in the following sections. The velocity and angulardistribution variations may be interrogated by electron ionization ofthe gas components (103) or after switching out the retarding potential(14) for releasing of the product ions after their transformations(104). Also it may be controlled by the timing the ion insertion intothe acceleration region. Ions with larger velocity (101) would fill alonger path than that of slower ions (102) during the insertion time ofions into the field free acceleration region (105). The region (106)ideally is field-free all the time even during the application of anorthogonal extraction pulse to accelerate ions through section 105 andonto the detector. Thus fast ions would be recorded over a wider spatialrange (111) on the multianode detector (107) than slow ions (112). Ifthese ions are uniformly distributed in time of arrival the numbershitting the first and the last anodes and relative intensities recordedat these boundary anodes in comparison with internal ones may give agood estimation for velocity of ions. For non-uniform distribution ofions along their path, which is usual the case for ion mobilityseparated ions, it is possible to shift the ion package as a whole alongthe insertion path of the orthogonal extraction region (105) by delayingthe start time of TOF acceleration relative to when the constantduration of insertion is begun. The shift of location of data inrecording line (107) would give the velocity of ions as a function ofthis time shift. Normally (when no DC fields are created in the finalpart of ion guide) the velocity of ions should be close to that of thegas flow. When accelerating or retarding fields like (76)—FIG. 7 or(24)—FIG. 1 are applied to the ion the measured velocity is directlyconnected with the temperature of cluster ions (it is proportional tothe square of this velocity difference) which thus provides anopportunity to estimate kinetic parameters for the decomposition ofcluster ions. This possibility is more directly applicable when iontransformations are performed on ions trapped in the middle of the ionguide. In the frames of the simple model of independent attachment ofatoms or molecules to some sites on the ion it is possible to decomposethe observed intensity distributions of cluster ions into the sets ofprobabilities of attachment to these sites. Such procedure may beperformed also for other processes like H/D exchange or protonation anddeprotonation of multicharged ions. For the problems for deconvolutionof the distributions of multicharged ions and for H/D exchange massspectra processing, this possibility was previously demonstrated in theart (see, M. O. Raznikova, V. V. Raznikov: “Protonation ProbabilityEstimation of Amino-Acids in Peptides and Proteins by their ElectrosprayMass Spectra” Chimicheskaya fizika, v. 20, N4, c. 13-17, 2001 (inRussian); M. O. Raznikova, V. V. Raznikov: “Determination of the extentof activity of H-atoms in ions of polyfunctional compounds by H/Dexchange mass spectra” Chimicheskaya fizika, v. 24, N1, c. 3, 2005 (inRussian)). Plotting the probabilities of attachment on a logarithmicscale as a function of inverse temperature will give estimations for theenergy of atom or molecule attachment (as well as activation energy ofH/D exchange or proton affinity) for each site in the ion and thefrequency factor. Independent and quasi simultaneous measurements of gasflow velocity are provided by recording of gaseous ions (103) producedby electron impact ionization (20)-(21), having a rectangular peak shapedue to the shape electron pulse (22). Location of the electron impactproduces ions (103) on the detector area (113) depends on this velocity,length of ion path, time shift between electron pulse and extraction ofions in TOFMS, m/z value of ions, and the TOF acceleration voltage.Among these ions (103) all neutral admixtures to the gas flow may berecorded including neutral products of decomposition of isolated ions.The latter may also be produced mainly by charge exchange between He⁺ions and neutrals which have much less ionization potential. Such ionsmay be valuable for providing reliable structure information. Forrecording of negative multicharged ions an attachment of He⁺ ions mayresult in dissociation of these ions similar to electron attachmentdissociation of multicharged positive ions. Ions coming after switchingof retarding potential (14) (FIG. 9) should produce a narrow bell-shaped(close to Gaussian) peak whose location (114) on the recording plane isconnected with collision cross section of ions and their mass andcharge. Thus overlapping ion peaks would be resolved by measurements ofion collision cross sections illustrated in FIG. 7 Electron attachmentdissociation (or laser dissociaton) of these ions may be additionallyvaluable in this case as it would give a set of ions with different m/zvalues which would simultaneously give structural information whilestill allowing to estimate ion velocity by comparison of locations ofthese ions (114) (fragment) and (115) (parent) the in recording plane.The shift between them is equal to the product of this velocity and thedifference in ion drift times in TOFMS. It is possible also in somecases to use multichannel position sensitive recording to estimate thedivergence of ion beams which directly gives the translationaltemperature provided that the corresponding axial velocity was measuredbefore. For this it is necessary for the divergence of the mobilitybuffer gas beam to be more than the width of anodes (107). Alternatelyshifting mass or mobility resolved ion packets from successiveinsertions into the oTOFMS region (105) between the first anodes and thelast anodes by appropriate delay of acceleration start relative to thestart of ion insertion would give information about divergence of theions. This shifting technique of course becomes unnecessary if eachanode is connected to its own analog and timing measurement channel in afast position sensitive detector. This measurement is simpler for lightions such as of He, Ar, Xe, but it is possible to do for large ions tooparticularly when a PSD of high spatial resolution is employed. This isimportant in the case where the large ions are heated due to some fieldsat the end of RFQ so their divergence would increase proportionally tothe square root of the temperature of heating.

The new method of ion heating herein described by using heavy admixtureatom impaction in the gas flow has the same advantage of direct heatingof the gas compared to heating of ions via moving them by electricfield. It is expected in the present case that ion temperatures would befairly uniform for all trapped ions independent of their structure andcharge. Thus it may be possible to use the process of charge exchangefor multicharged ions (peptide and protein, for example) to provideinformation about proton affinities, averaged local electric fieldinside the ion, and for formation of clusters. FIG. 11 illustrates theidea. Schematically shown multicharged ion (120), (peptides forexample), have several possible locations for positive charges (additionof protons for basic residues) and negative charges (removal of protonsfrom acidic residues). If an admixture molecule with large protonaffinity (ammonia molecule 124 is shown as an example) comes close tosome proton location (123) and this molecule has a chance to capture theproton, then the effective charge of the ion is reduced from 3 to 2. Onthe other hand, an admixture molecule with a weakly bounded proton(formic acid is shown—125) can attack a negatively charged site (126)where it increases the peptide ion charge from 3 to 4. After theseproton transfer reactions, formation of positively charged NH_(4.) ⁺ ionand negatively charged HCO⁻ ₂ on are possible. To easily remove theseproduct ions from the multicharged ion it is possible to use an excessadmixture of polar molecules in the gas flow (like water (127), forexample) to cluster with the ion. Thus the small ions would beassociated with water molecules and may be removed from this largecluster ion more easily. The other possibilities for removing ionizedsmall admixture ions from the peptide charge transfer site is totransfer the admixture ion protons to water molecules to form H₃O⁺ ions(128) or to draw protons from water molecules to produce OH⁻ ions (129).These water ions themselves may provide charge exchange processes withthe bio-ion as shown in FIG. 11 as illustrated with the use oftwo-headed arrows. The probabilities of such events are dependent bothon ion temperature and on the difference between proton affinities ofthe attacking molecule (or ion) and the corresponding ion site.Separation of the charged species to “infinity” in principle give theproton affinity of the ion site (123, for example) but in fact shouldalso include a contribution of local electric field potential created byother positive charges of the ion (121) and the negative ones (122) and(126), shown by arrows. The proton affinity of the corresponding residuein the ion without influence of a local electric field may be estimatedby measuring before proton affinities for corresponding small singlecharged ions. For a large enough solvent water cluster shell around thebio-ion and relatively small concentration of admixtures of basic andacidic molecules in the gas flow the charge exchange processes with thebio-ion should be provided mainly by water ions (as occurs in-vivoaqueous solutions wherein the main factor controlling this chargeexchange process is the pH-value). To estimate the pH-value in our caseit is sufficient to record the corresponding cluster ions and calculatetheir average composition. It is possible to switching out the clusterwhich have formed in the gas flow so that the composition of theclusters would also be “frozen” so as to conserve the steady stateequilibrium distribution. To accomplish this, it is necessary to takeinto account that molecules of water, ammonia and formic acid havesignificantly more energy than helium atoms and corresponding energy ofthermal oscillation of the ion so their impact on the biomolecule insteady state conditions would provoke on average some evaporation of thecorresponding molecule. Spontaneous evaporation (exponentially dependenton ion temperature) can also take place but it should be significantlyless. Thus if we reduce proportionally the concentrations of theconsidered admixtures and conserve the temperature of the ion (by acorresponding increasing of Ar admixture) up to the moment when theseadmixtures disappear totally and after that to quickly switch out the Arflow, then the steady state composition of ion clusters should beconserved with good precision. Probabilities of charge retention bydifferent sites of the ion can be calculated via intensity distributionsof multicharged ions by the method already mentioned (M. O. Raznikova,V. V. Raznikov: “Protonation probability estimation of amino-acids inpeptides and proteins by their electrospray mass spectra” Chimicheskayafizika, v. 20, N4, c. 13-17, 2001 (in Russian)). For small temperatureintervals, logarithmic plots of the multicharged ion intensities as afunction of the inverse ion temperature would allow calculatoinalestimates of the proton affinities of the corresponding sites. Anotherpossibility is to use these estimations of proton affinities forpredicting conditions for creating a desired value of the average ioncharge. This may be important to perform additional separation ofisolated ions similar to the isoelectric separation of biopolymers inliquid and gel solutions. For high enough ion temperatures thetransitions between different ion charge states as well as betweendifferent compositions of the clusters under the given flow of water,basic and acidic molecules would be fast enough. Thus it is possible toconsider that bio-ions slowly moving in the gas flow and under electricfields in the RFQ isolation region have a constant charge and crosssection which is equal to its average charge and cross section ofclusters. This is the same assumption forming the basis of isoelectricbiopolymer separation in liquids and gels which results in significantresolving power of this method compared to the case when ions with fixedcharges are moving. This is true in our case too. For example, a 10%difference in average charge may be less than one charge for smallcharged ions but it provides a 10% difference in mobility of ions whichare large and these ions may be easily separated by applying in the gasflow an appropriate retarding potential which can stop more highlycharged ions but release ions with less average charge. It may beexpected that resolving power of this separation would increase withdecreasing of the average ion charge. Simulations show that large enoughions in a physically reasonable RFQ can be separated if they havemobility difference of about 1%. After isolation of some multichargedions (or ions which are expected to have many sites to be charged) manymanipulations can be performed with and on theses isolated ions. It maybe useful, for example, to try to first determine the maximum possiblecharge number for the given ions. For determining maximum positive ionscharge states a concentrated flow of acidic molecules combined withwater molecules should be directed onto the ions while the temperatureis regulated, perhaps, by some additional flow of Ar atoms. After that,all admixtures to the gas flow are removed, the retarding potential isswitched out and ions are recorded. If necessary, the cluster shell maybe removed by strong enough heating by Ar admixture flow. In cases wherewe observe ions with different numbers of charge it may mean that eithersteady state conditions are not achieved or that the isolated ions donot correspond to the same molecule. In case fragment ions are observedit means that for the temperature used such multicharged ions,unfortunately, are not stable. In this case complimentary ion pairs maybe found: two ions with sum of masses being equal to the expected massof the ion. In the event that some of these pairs possess differentnumber of total charges than the isolated ions this may indicate alsothat a non-uniform or steady state conditions are not achieved. It ispossible to try to estimate the number of sites in the ion wherenegative charges may be located. For example, for peptides and proteinsusual sites for negative charges are acidic residues: glutamic acid,aspartic acid and also acidic group at the C-end of the molecule. Thesesites may be “closed” in a sample of control molecules by specificchemical reaction via formation of esters by some admixture moleculesadded in the buffer gas flow such as methanol, for example. In this caseion composition would be changed by some substitutions of OH groups byCH₃ groups. In case several ions with different number of suchsubstitutions are observed the isolated ions may be also non-uniform orsteady state conditions are not achieved. Kinetics of these processes(in case some water admixture is added also to the gas flow for havingreverse reaction—hydrolysis of ester bonds) may be investigated usingthe same approach, mentioned above for decomposition of intensitydistributions of ions with different number of such substitutions.Probably, in this case some conclusions about the types of acidic sitesin the ion and influence of the ion environments for these sites may bemade. Determination of the number of positive and negative charges sitesis essential information for information about proton affinities ofdifferent sites in the molecule as well as an averaged internal electricfield in the ion. Additional separation of isolated ions may beperformed, for example, in the following way. At first the retardingpotential is adjusted to stop all these ions with average charge morethan 1 and release singly charged ions or with less average charge. Incase of measuring of multicharged positive ions the relativeconcentration of basic molecules (may be stronger than ammonia) in theflow is increased gradually up to the moment of recording the first twoexpected single or more charged ions. Two ions are necessary to recordto estimate the time when all isolated ions may be released in case theyhave the same average charge. After that stronger retarding potentialfor stopping ions with average charge significantly less than 1 (0.1,for example) as shown in FIG. 3 (41) is switched on. Thus low chargedions would be accumulated in a new position and if some of isolated ionshave enough extra average charge they would stay at old place. Aftersome time sufficient for shifting of “all” isolated ions to a newposition the rest of the ions (if any) which are located before thefirst retarding potential are removed by a corresponding rotating fieldto rods or they may be conserved for further measurements by gentleexcitation with a sum of rotational fields resonant for each ion chargepossible for isolated ions. So ions which have just been so selected maybe investigated separately from their neighbors from the previousisolation. After releasing these ions or their products oftransformation the remaining ions which are just rotating may bereturned into the gas flow by switching out of rotation field and theymay be subjected to further separation and all other procedures what wasdone with the first portion of selected ions.

In some cases it is necessary to further select the ions. Non-uniformityof selected ions may be understood by recording of their mass spectrawhich reducing the retarding potential in conditions when flowadmixtures remain constant. To maintain high enough temperature of theions to prevent them from forming too large clusters a decreasingretarding electric field along RFQ would be created to provide close toconstant drift velocity of the ions as it was described previously inthe sections devoted to the measurements of cross-sections of the ions.The difference here is that ions during their drift in RFQ would besubjected to relatively fast steady state transitions between differentcharge states and compositions of the cluster shell. Thus the measuredaverage velocity of such ions by multichannel data recording asdescribed in the previous sections would correspond to some average ionformed in conditions slightly different from those for the region ofisolation. The main difference in our case would be a somewhat morerelative steady state concentration of acidic molecules as they haveless angular divergence than other admixtures; thus their approach tosteady state conditions in the gas flow would occur more slowly than forother admixtures. In case to conserve as far as possible the conditionsof the cluster formations it would be possible to get an upperestimation of the average mobility of primary ions selected andsubsequently released by reduced retarding field. At first ions havinglow charged and larger cross section ions would be released. Noticeablydifferent average m/z values of the ions or significant difference oftheir average velocities for different releasing retarding fields wouldindicate that these ions are not uniform. Using rotating fieldexcitation of the drifting ions may allow the removal of undesired ionsfrom the flow and estimate the average m/z value of ions of the interestin the flow. It would give a low estimation of average m/z of these ionsin the region of isolation and it may be used for additional separationof desired ions. The possibility of this separation is based ondifferent distribution of different admixture molecules across the gasflow on some distance from the exit tube as it is shown qualitatively inFIG. 12. Molecules such as formic acid (131) which are heavier thanammonia would have about 1.64 times less divergence than that of ammoniamolecules (133) which is about twice less than the divergence of He(130). The water molecules having molecular weight close to that ofammonia would have almost the same divergence. Thus at some distancefrom the flow axis the relative concentration of ammonia would rapidlyincrease in comparison to that of formic acid, therefore ions shiftedfrom the axis (137) would have significantly less average charge thanthose near the axis of the flow. Ions would restore their previousaverage charge after returning onto the axis to the place (134) wherethe retarding electrical force (136) is compensated by the draggingforce of the gas flow (136). The compensation may also be done byapplying a sum of rotating fields to selected ions located in specificregions along the RFM. To select the desired ions the interval ofrotating frequencies should correspond to the interval of m/z valuesstarting at an m/z slightly below the estimated average m/z ratio of thedesired ions which are in the flow and up to slightly beyond this ratiowith z being equal, for example, to 0.1. The amplitudes of the rotatingfield are increase so that some ions would move off of the axis to adistance determined by their having an average charge of 0.1 (138).Thus, the ions could overcome a strong retarding potential, (which canbe produced in this case by relatively small retarding force (139) whichis less than the dragging force (140) of the gas flow) and wouldtherefore come out of the rotation region, return to the flow axis (141)and then increase their average charge. They can then be recorded oralternatively isolated for further investigations. The isolation of theions is not very dependent on momentary increases of the rotating fieldamplitude or frequency occasional increase of their rotation radiusbeyond that defined by their average charge of 0.1 would result incommensurate reduction of the average ion charge which then causes theions to moves from conditions of resonant rotation and reduces theirrotating radius to the region where the average charge is 0.1. Bymeasuring m/z values and the velocities of ions as they are beinggradually released, the appropriate amplitude of the rotating fieldwhich is suitable for releasing the desired ions may be determined.Switching on the additional retarding potential at the moment thedesired ions start to be detected in the mass spectrometer and stoppingany further increase in the rotating field would allow isolation of thedesired ions for further investigations.

The generation of rotating fields for a set of chosen frequencies andamplitudes may be important from another point of view as well. Massspectrometry and IM/Mass Spectrometry. For example, such known problemof multicharged deconvolution of electrospray mass spectra may be areally difficult problem for investigation of heavy mixture of bio-ions.Usual mobility separation in this case may even complicate the problem.It may separate ions corresponding to the same biomolecule having thesame number of charges but different cross sections due to differentdistribution of charges inside the ion. From that point of view, fastrandomization of charge distribution inside the ion during the processof mobility separation may be important for real separation of differentbiomolecules. Thus, it may be advantageous to first trap ions from theinitial gas flow not only ions for chosen m/z and mobility values butall ions with chosen mass with all possible charge numbers and within arange of expected mobility values for each possible charge number. Suchtrapping may be provided by switching on a predetermined sum of harmonicrotating fields with given set of frequencies and amplitudes in the wayas shown in FIG. 5 during the ion flow. Furthermore, all these trappedions are inserted into the gas flow by switching out the rotating fieldand are then accumulated at some position inside the flow by applyingstrong and short duration retarding potential. By the procedures justdescribed ions may be separated, using controllable flows of water,basic and acidic molecules, on the basis of their different abilities toobtain and lose charges and form clusters in such flows. This separationwould be a real separation of biomolecules and not a separation ofdifferent ionic forms of the same biomolecule as may occur in the usualpractice of mass spectrometry and ion mobility separations. In the caseof large biopolymers investigations where the biopolymer has multiplesites for both positive and negative charges it may be possible to use asimpler method of separation based on providing conditions close to theisoelectric point for the molecule of interest. The idea is to providesuch conditions at the region close to the beginning of RFQ (154) inFIG. 13, where the gas flow is not yet diverged significantly and therelative densities of admixtures near the flow axis: (5)—Ar,(151)—formic acid, (152)—ammonia, and (153)—water are close to those atthe beginning of the supersonic flow. Switching on two strong shortretarding potentials: (159) to stop positive ions (154) and the next(160) to stop negative ones (155), would allow only species with averagezero charge (156) to come through them. These particles moving with thegas flow would come to the region where due to different divergence ofacidic and basic admixtures the relative density of acidic molecules ismore than that in the region of retarding potentials and the consideredparticles would became gradually preferably positive (157). Maximumincrease of this relative density in case of formic acid and ammonia is˜2.7. In case it is enough to provide average charge of the molecule ofabout 1 the corresponding ions (158) would be recorded with applicableefficiency. It is important that molecules which have an average zerocharge do not come out of the gas flow before their charge issufficiently increased. Negative charging of these molecules wouldresult as they came to the edge of the gas flow since the relativedensity of ammonia is larger here than near the axis of the flow.Therefore these molecules with average negative charge would be returnedto the gas flow by RF-focusing of the quadrupole.

Alternatively it is possible to provide conditions close to theisoelectric point of the molecules of interest in the region ratherclose to the end of RFQ where gas flow composition near the axis isclose to a steady state situation. In this case the average charge ofthe ions which could pass through positive and negative potentialbarriers like (159) and (160) in FIG. 13 would be close to zero up tothe exit (18) from RFQ. On the other hand the gas flow density is lowenough in this region so that the average time between collisions of theions with gas molecules is much less than that at the beginning of theRFQ. Thus the average frequency of ion transitions between differentcharge states (provoked by collisions) may be not so large andsignificantly less than the frequency of RF-field (few MHz usually). Itmeans that ions with average zero charge but consisting at each momentof some mixture of positive, negative ions and zwitter-ions would befocused to the axis of RFQ as both positive and negative ions areinfluenced effectively by the same force directed to the axis andzwitter-ions could be transformed to usual (not zero charged) ions aftersome time (hopefully less than the time of these ions traveling to theend of RFQ). Thus the diffusion of these ions may be significantly lessthan that for the real neutral particles and its part which could comeout of RFQ may be close to those for usual ions. No DC fields areapplied in this case at the end of RFQ as they are not very effective inthis case. However, it may be reasonable to apply some voltages betweenRFQ and the exit aperture like (16) and (17) shown in FIG. 1. Ions inthis region would not have much possibilities to change their chargestate which means that positive ions would be accelerated (by the fieldsas they are shown), the corresponding negative ions would bedecelerated, and zwitter-ions would conserve their velocity. Thisproperty may be used further to distinguish the origin of recorded ionsfrom initially positive, negative or from zwitterions. To preventsignificant cluster formation a short path for the ions between theplace of isolation and the exit orifice of RFQ region (18) is necessaryso as not to allow the average ion velocity to come very close to thegas flow velocity (and thus for ions not to be excessively cooled). Thisaverage ion velocity for a given experimental conditions would bedependent on the averaged ratio of mass to collision cross section ofions and could be measured by multichannel recording in the TOFMS. Torecord zwitterions they should be converted to charged particles. It maybe done, for example, by electron attachment to some positively chargedsite of a zwitterion if the pulse (77) shown in FIG. 7 is applied toelectron impact ionizer anode (21). Also it is possible to do byproducing of He⁺ ions by electron impact inside the gas beam (19) incase the anode pulse has a view (22) shown in FIG. 1 and attachment ofthese ions to some negatively charged sites of the zwitterions. It maybe reasonable also to decompose zwitterions by laser beam to producepairs of positive and negative ions. In case it is done insideextraction region of TOFMS it could be possible to detect this pair inco-incidence since their parent ion is the zwitterion, therefore morereliable structural information may be measured in this case.

To record such ion pairs as well as uncorrelated positive and negativeions, a Bipolar Time-Of-Flight Mass Spectrometer (BiTOFMS) shownschematically in FIG. 14. It is a simplified linear BiTOFMS. Reflectrontype instruments of this kind are possible as well with both parts forpositive and negative ions having tilted geometry. Ions (170) aredirectly inserted from exit orifice (18) FIG. 13 into the middle ofacceleration region where electric field (184) is created for the timeof ion extraction. Positive ions should be accelerated to the bottommultianode recording plane (175), negative ions—to the top one (176).The field “free” region for positive ions (172) is about twice as longas the acceleration region (171) for them, as well as this relation isvalid for negative ions: the field length (173) is twice less than thefield free gap (174). Between inserted ions some zwitterion (179) isshown at the beginning of its travel inside the BiTOFMS with someinitial velocity (179). At some chosen time after the start ofextraction of usual positive and negative ions (not to confuse them withthe products of the zwitterions) under influence of laser beam (177) thezwitterion decomposes into positive (180) and negative (181) ions. Intheir center of mass reference system these ions have oppositevelocities ((182) and (183)) whose magnitude is inversely proportionalto the ion masses. These velocities are added to initial velocity (179)to give start velocities of ions for their acceleration in the field(184). According to their m/z values, start position, and startvelocities they are recorded in due times by the corresponding channelsof both recording systems of the BiTOFMS. As velocities like (179)should be close to the average velocity for all ions produced from thesame parent molecules their estimation may be received by comparison ofrecording positions of ions of different m/z values as it was describedearlier herein devoted to the measurement of ion cross sections.Comparing of recording positions of ions (180) and (181) for measuredtimes of their motion to recording plates (175) and (176) would givesome estimations for the axial components of velocities (182) and (183).As these velocities (and the axial components as well) should beinversely proportional to the ion masses (or their m/z values estimatedfrom the times of recording) it gives a means for confirmation thatthese ions are produced from the same zwitterion. The sum of masses ofthese two ions would give the mass of the zwitterion (including somecluster shell). Comparison with “exact” mass of the molecule itself ifit was measured before would give the mass and the composition of thisshell and some estimation of orthogonal to recording planes componentsof ion velocities (182) and (183). Using these estimations for a numberof such zwitterion decompositions it may be possible for known photonenergy of the laser light to evaluate the effective strength of thecorresponding bond which would include the average energy ofelectrostatic attraction of the separating ions (180) and (181) andprobably the energy spent for removing of some cluster shell molecules.Having this information for different pairs of these ions someconclusions about zwitterion conformation could be done. Other mentionedways of converting of zwitterions into recordable ions have no suchclear possibilities. On the other hand in these cases it is possible toprovide less divergence of the ion beam (170) and to have betterresolution and accuracy of m/z measurements. As recordable ions areproduced just after the RFQ exit it is possible to reflect them by aparabolic (or a proper cylindrical) mirror to convert a divergent ionbeam into quasi-parallel one as it is described in co-pending U.S.patent application Ser. No. 11/441/766, filed May 26, 2006, for unipolarions. However, in a case of bipolar ion flow, the mirror with a chargedthin dielectric film described in U.S. patent application Ser. No.11/441/766 will not be optimal. However, reflection of bipolar ions ispossible from a surface polarized by alternating charges. This effect issimilar to ion focusing by RF-field—ions moving quickly along a surfacewith changing potentials would be actually influenced by some fastalternating field. However, estimations show that randomly distributedpositive and negative charges on some surface due to high enough averagefrequency of this field would not provide a strong enough repulsionforce for ions having the energy (velocity) of motion orthogonal to thesurface which is expected in our instrument. On the other hand, it ispossible to fabricate effective ion mirror surfaces from thin layers ofconductor and dielectric and to connect adjacent conductors to bipolarvoltages of the same absolute values. Also it is possible to fabricatesuch structures from piezoelectric thin film structures with patternedelectrodes. Ions in this case should move in directions close to theorthogonal to one to the layers. For velocities of ions of about 1000m/sec (close to the velocity of helium gas flow in our case) thethickness between the layers of few tenths of mm would be suitable as itwould be equivalent of the frequency of effective AC field of few MHz.Low voltages applied between these layers would be enough to reflectmost ions and the voltages can be empirically optimized for ions ofspecific m/z. Thus it is also possible to increase the voltage on theselayers in time so that the best field strength is present to reflect themobility selected ions which are eluting from the cell at that time. Ifnecessary, significantly stronger voltages may be applied withoutcreating a glow discharge since the gas pressure in the region of theirusage is low enough. FIG. 15 illustrates this idea. Positive (190) andnegative (191) ions come to parabolic or cylindrical mirror (192). Thefocus of this mirror is located in the center of exit orifice (18) ofRFQ shown in FIG. 13, for example. The mirror may be built from isolatedconductor layers to which positive (193) and the same value negative(194) voltages are applied. Ions coming out of the orifice (18) arereflected from this mirror and form a quasi-parallel ion beam. This beamis reflected from the flat mirror (195) built in the same fashion as theparabolic mirror; from isolated conductor layers with positive (197) andnegative (196) voltages applied to them. After this reflection positive(190) and negative (191) ions are then simultaneously inserted into theoTOFMS simultaneously measured by applying a pulsed field (184) whichsimultaneously accelerates them to the positive (175) and negative (176)multi-anode detector recording planes. Using more complicated system ofmirrors, for example, one parabolic and two flat mirrors may allow oneto recreate the same direction of the ion beam axis as that prior to themirror system. Also, mirrors can be constructed with different shapesand combinations which we suggest as an alternative which can providepoint to parallel focusing or point to point focusing using speciallydesigned mirrors. An internal surface of ellipsoid of revolution is ableto collect beams coming from one focus and project the beam into the asecond focus. It is possible of course to coat some part of this surfaceby thin dielectric film which could be charged with one ion sign, butthe more general way to construct such devices is with the interleavedelectrodes in analogy to the mirrors shown in FIG. 15 by assembling ofkapton foil electrode sections. An Alternative way to focus ions to theinput orifice of an MS is to use this flexible circuit for producing ofboth mirrors in which their shapes can be obtained by constructingcorresponding ellipsoids and cones having the desired focuses to providefocusing ions from the aperture (18) to the MS input orifice by point topoint focusing. This is not useful for an oTOF instrument, but can bequite useful for any other mass spectrometer which does not depend onparallel ion beams. A cylindrical ellipsoid may be replaced bycorresponding osculating circles without noticeable losses in focusingproperties. In cases when additional differential pumping stages arenecessary the point to point focusing mirror systems may be repeated anydesired number of times. It is also possible to then replace the lastset of elliptical mirrors with the corresponding parabolic mirrors ofFIG. 15 which will then re-establish a quasi-parallel ion beam totransport ions without noticeable divergence for relatively longdistance for insertion into an oTOFMS or into the trapping region ofinstruments such as an ICR or “Orbitrap”.

In cases when isoelectric selection of biomolecules described in theprevious section fail to provide pure isolation of the desiredbiomolecules, it is possible to combine it with previously describedseparation methods. Between a variety of possible versions of suchcombining it is possible, for example, to provide separation ofbiomolecules by two isoelectric points specific for the desired compoundfor two different experimental conditions. These conditions may includebesides different temperatures some admixtures which may influence oncharge exchange processes for biomolecules, for example, containingalkali metals which ions can substitute proton and compete with it foroccupation of corresponding sites in the biomolecule. It is possible toprovide the first isoelectric separation at the beginning of the ionguide as shown in FIG. 13 and to stop just separated ions by similarpair of retarding fields (159), (160) as shown in this figure butlocated at the end of the ion guide where these ions would acquire somenonzero average charge. By then changing the composition of the gas flowthe condition for the second isoelectric separation are provided andions with average zero charge are recorded as described in the previoussection. Hopefully it would be enough to receive the flow of ions of thedesired compound without noticeable admixtures. Otherwise morecomplicated schemes of multistage separation could be performed. Some ofthe methods described by Vandekerckhove, et al. in 2005 (U.S. Pat. No.6,908,740) for liquid phase specific chemical and/or enzymaticalteration of selected types of peptides which are suitable for gasphase implementation may be used with the present invention. In casethese alterations provide significant change in mass of some peptidesadditional separation may be achieved by resonant rotating fieldexcitation of ions. If charging properties of some molecules are changedisoelectric separation may give different results comparing to thosewithout any alterations.

The bipolar TOFMS and new approaches for isolation of desiredbiomolecules described in previous sections and the concept ofsimultaneous measurement of positive and negative ions from the samesample (disclosed in co-pending U.S. patent application Ser. Nos.11/441,766 and 11/441,768) may be implemented in another way asdescribed below. Instead of moving of positive and negative ions fromthe trapping region in opposite direction to different TOFMS instrumentsdedicated for separate recording of positive and negative ions, thepositive and negative ions together are directed to the single bipolarTOFMS To accomplish this, RF-voltages are decreasing along the entrancemobility tubes and are applied to the sections of these tubes. Ions ofboth sign would be pushed in direction of decreasing voltages and forstrong enough gradients, this would overcome the counter gas flow comingfrom these entrance tubes. For a narrow range of m/z values andmobilities a constant set of these RF-voltages may be applied allowingtransport of the ions inot the BiTOFMS. In this case this system maywork in “continuous” mode. This separation may be not so important incase of analysis of mixtures of multicharged bio ions as they may be forisolating by rotating field on the base of their m/z values and furtherseparation on the basis of their isoelectric points as described before.However, analysis of complex bioion mixtures with broad distribution ofions in their m/z values and mobilities could not be done effectivelyfor a single set of RF-voltages. In this case smaller values andgradients of these voltages may be used for relatively small ionswhereas larger values and larger gradients (or reduced frequencies ofRF-voltages) may be used for larger ions. Also for some cases additionalseparation of the ions by their travel time from the trapping region toTOFMS may be useful for some kind of measurements. In this case in orderto provide a “pulse” mode measurements, an RF-voltage plug appliedbefore the trapping region may be switched on for the time of ioninsertion into the entrance tubes and so prevent the next portions ofions from coming into the trapping region which would spoil the previousion separation. As ion decomposition as well as other iontransformations may be performed under controllable conditions insidethe RF-quadrupole CID (Collision Induced Dissociation) tubes may beomitted and the motion of ions of both signs inside the exit tubes maybe provided by some gradient of RF-voltages applied to the sections ofthese tubes. Focusing of ions and their rotation excitation are providedindependently of the ion sign with rotating of ions of different signssimply having a phase shift being equal to π or 180°. Stopping of ionsof both signs inside RFQ may be provided by corresponding RF-voltageplugs applied between adjacent sections of RFQ rods.

Previously described isolation of ions by using a resonant rotationfield may be performed with less efficiency and resolution in case ofabsence of supersonic gas flow inside RFQ. In this case DC, RF or ACvoltage plugs should be adjusted to stop ions rotating close to rods andallow travel along the RFQ axis for non-resonant ions under influence ofsmall longitudinal electric field. Addition, in necessary cases, ofheavy admixtures and an increase of the rotation field would result inheating of ions and in their decomposition. Ion-fragments can come outof resonance of rotating field and come to the RFQ axis. By use of anappropriate voltage plug it is possible to stop these ions near the axisand after finishing of decomposing of primary ions by correspondingrotating excitation. It is thus possible to select the desired productions for further decomposition. The remaining ions may be transported toTOFMS. This procedure may be repeated the desired number of times. Thus,MS^(n) technique may be implemented in the present invention also.

An important advantage of the method and apparatus disclosed above isthe isolation of ions and their transformation since it is a possible toinitially trap a chosen set of ion types and then investigation themeach in turn. By contrast, conventional methods usually can onlyinvestigate ions of one type (which are subjected for a given time tocollision induced dissociation, for example) during which time all otherinitial ions are lost. To overcome this limitation Loboda, et.al. in2005, US Patent Application 20050253064 A1, suggested a method of massselective axial ejection of ions from linear ion trap by changing of iondragging forces provided by DC and AC fields. However, this approachincludes as a first step trapping of all ions produced in previous stageof the system. It is desirable also to provide a possibility of furtherinvestigations of product ions received at least from some types of theprimary trapped ions. Unfortunately, trapping of the product ions inthis case (as it is described above) may be complicated when m/z ofthese ions are close to those for some primary trapped ions as such ionsmay be confused in further steps of investigation. To solve this problemit is possible to use instead of a single RFQ ion guide with supersonicgas flow some sequence of such ion guides when the first ion guideaccepts ion flow from a mobility cell or directly from some ion sourceand the last one delivers ions into a TOFMS. Schematic of connectionbetween a pair of these adjacent guides is shown in FIG. 16. The productions (201) and (202) produced in the left ion guide (200) are movingunder influence of the supersonic flow inside this guide of helium atoms(8) with possible admixtures such as argon (5) and possible electricfield along RFQ axis (203). Under optional focusing field (205) theseions (207) come out of this guide and overcome the counter helium flow(208) from the right ion guide under possible help of electric field(219) between focusing electrodes (206) and entrance plate of the rightion guide. The main part of initial helium flow (210) inserted into theright ion guide is directed into sectioned tube (212) where as forprevious ion guides some admixtures like argon (5) and xenon (6) can beinserted into the flow. The gas coming from the tube (212) turns into asupersonic gas flow. Under the influence of corresponding rotatingfields the desired product ions (214) are trapped at the beginning ofthe RFQ (211). They are stopped inside RFQ by electric field (215) asdescribed before. After trapping of desired product ions is finished thetrapped ions may in turn be moved to a place of further transformation(216) provided by a corresponding electric field (217). Their productsmay then be trapped and investigated in additional ion guides as justdescribed. Such operations may be repeated a desired number of timesrestricted only by the number of the ion guides connected in seriesbefore the TOFMS. To provide the necessary conditions for the desiredgas flows the corresponding pumps such as (204), (209) and (218) shouldbe used in the system.

Additional capabilities for getting structural information about largeenough bio-ions may be provided by somewhat the more sophisticatedoperation of an electron impact ion source located after the exitorifice of the RFQ ion guide than that described before. A simplifiedschematic of this ion source with indirect heated cathode (75) is shownin FIG. 7. Using as cathode material of lanthanum hexaboride crystalallows one to have a large enough density of electron current (few mAper square cm) and relatively low width of electron energy distribution(close to 0.1 eV). Simple estimations show that for cathode length ofabout 1 cm, a large bio-ion with collision cross section of about 1000Å² moving with helium flow across a continuous electron beam withcurrent density 2 mA/cm² would be impacted by an electron withprobability close to 0.1. The probability is large for observingproducts produced by two or more electron impacts. The expected resultof such impact with electron with energy of few eV (less than ionizationpotential of the biomolecule—typically close to 10 eV) would bedissociation of some bond in the molecule probably close to location ofthe electron impact. To provide this dissociation the energy of electronimpact should not be less than the corresponding bond strength. Byrecording of yields of ions produced by dissociation of some bonds inthe bio-ion for different electron energies after processing of thusreceived ion appearance curves it would be possible to get seemingthresholds for the corresponding bond dissociations. The procedure ofprocessing may be close to that described in our old paper devoted toprocessing of ionization efficiency curves for some simple molecules andradicals (Raznikov, et. al. Int. J. Mass Spectrom. Ion Proc. v. 71,1986, p. 1-27). Precision for ionization threshold determinationsnoticeably better than 0.1 eV was demonstrated in this work. It isnecessary to take into account that dissociation thresholds determinedin such a way for given bio-ion may deviate significantly from thecorresponding bond strengths. The main reason for that may be a changedkinetic energy of impacting electron in the local electric field of theion. For example on the distance of about 10 Å from location of positivecharge in the ion electron impact energy would be more than its initialenergy for about 1 eV (if polarization influence of atomic arrangementof the ion may be neglected). In case the corresponding energies of bonddissociations were determined before by ion heating under influence ofheavy admixtures to the helium flow as described already here thencomparison with electron impact thresholds would give some informationabout the distribution of local electric field inside the consideredbio-ion. It may give a possibility to draw important conclusions aboutthe space structure of bio-ions in gas phase which is not possible to doby other methods. Besides that when dissociation of some bond in thebio-ion produces not one but a pair of recordable ions the coincidenceof the corresponding appearance curves would give additionalconfirmation of the origin of the product ions from the same parent ionwith expected sum of these ion masses being equal to the mass of theparent ion. Alternatively to recording of product ions for differentelectron energies it is possible for given electron energy to change thetemperature of the ions by inserting of different retarding (oraccelerating) fields at the end of RFQ and/or changing contribution ofheavy atoms in the helium flow. Plotting of relative ion yields inlogarithmic scale for inverse ion temperature hopefully would showArrhenius type curves as for the described similar situation whenphotoionization of biomolecules was investigated (Wilson, et. al., J.Phys. Chem. A v. 110, 2006, pp. 2106-2113). Angle coefficients derivedfrom these curves would give the activation energies for thedissociation of corresponding bonds what usually is considered as a goodestimation of the bond strengths for pure thermal decomposition. For thecase of electron impact it may be expected that these activationenergies would be close to differences between the corresponding bondstrengths and the energy of electron in place of impact. Comparison ofresults of these kinetic measurements with estimations received viaprocessing of ion appearance curves under varied electron impact energymay give better understanding of ion decomposition processes occurringin the considered conditions.

The applications of this instrumentation and methods are not restrictedto analysis of the structure and mass of gas phase ions. This inventionmay also be used for isolation, cooling and selected area soft landingdepositions of mass and mobility selected ions. Also, as in analogy tocombining this instrument with a highly monochromatic electron impactionizer, the instrument can also be combined with other instruments andtechniques for ionization or known to those skilled in the art. Amongsome of these examples would be the use of the long trapping times andhigh mass capability, which would allow co-measurement of luminescencelifetimes and quantum yields while the mobility and mass-selected ionsare rotating in the trap. In particular this can be powerful when eitherthe intrinsic luminescence or the luminescence from a luminescence tag,including but not limited to aromatic molecules and lanthanide(especially Eu), which among other uses, are useful in determining watersolvation chemistries of isolated ions. Since ions with a solvationshell can be prepared and isolated and trapped, the co-application oftechniques such as circular dichroism, real-time x-ray scattering, andphotoelectron spectrometry using either standard excitation sources orsynchrotron light sources could provide extremely useful measurements ofthe structure of the trapped solvated and unsolvated gas phase ions in away which would indicate their probable in-vivo conformations.Photo-ioniztion and photo-fragmentation of ions within the rotationaltrapping regions or trapping regions within the dense gas flow are alsopossibilities.

Although the present invention and its advantages have been described indetail, it should be understood that various changes, substitutions andalterations can be made herein without departing from the invention asdefined by the appended claims. Moreover, the scope of the presentapplication is not intended to be limited to the particular embodimentsof the process, machine, manufacture, composition of matter, means,methods and steps described in the specification. As one will readilyappreciate from the disclosure, processes, machines, manufacture,compositions of matter, means, methods, or steps, presently existing orlater to be developed that perform substantially the same function orachieve substantially the same result as the corresponding embodimentsdescribed herein may be utilized. Accordingly, the appended claims areintended to include within their scope such processes, machines,manufacture, compositions of matter, means, methods, or steps.

1. An apparatus for the analysis of gaseous ions and neutral species andmixtures thereof, said apparatus comprising: an ion source for theproduction of gaseous ions and neutral species and mixtures thereof; agas flow formation region fluidly coupled to said ion source, said gasflow formation region comprising a sectioned capillary having at leasttwo electrodes, said gas flow formation region operable to accept ionsand/or neutral species from said ion source; at least one sectionedradio-frequency multipole ion guide fluidly coupled to said gas flowformation region, said sectioned ion guide comprising a plurality ofelectrically isolated electrode sections; an exit orifice fluidlycoupled to said at least one sectioned radio-frequency multipole ionguide; and, a detector fluidly coupled to said exit orifice.
 2. Theapparatus of claim 1, wherein said gas flow formation region comprisesan input orifice.
 3. The apparatus of claim 3, wherein said inputorifice is coupled to a gas or liquid delivery valve.
 4. The apparatusof claim 1, wherein said sectioned radio-frequency multipole ion guidecomprises mulitelectrode rods.
 5. The apparatus of claim 5, wherein saidmulitelectrode rods are separated by grounded plates isolated fromground.
 6. The apparatus of claim 1, said at least one sectionedradio-frequency multipole ion guide comprises a radio-frequencyquadrupole.
 7. The apparatus of claim 1, said at least one sectionedradio-frequency multipole ion guide comprises a radio-frequencyoctapole.
 8. The apparatus of claim 1, wherein said at least onesectioned radio-frequency multipole ion guide comprises a differentialpumping region.
 9. The apparatus of claim 1, wherein said electricallyisolated electrode sections are coupled to a component selected from thegroup consisting of a DC voltage source, an AC voltage source, a RFvoltage source, and any combination thereof.
 10. The apparatus of claim9, wherein said voltage sources are coupled to, and controlled by, acomputer.
 11. The apparatus of claim 1, wherein said gas flow formationregion is a supersonic gas flow formation region.
 12. The apparatus ofclaim 1, wherein said detector is a mass spectrometer.
 13. The apparatusof claim 12, wherein said mass spectrometer is an orthogonaltime-of-flight mass spectrometer.
 14. The apparatus of claim 13, whereinsaid orthogonal time-of-flight mass spectrometer comprises a positionsensitive multi-anode detector.
 15. The apparatus of claim 13, whereinsaid orthogonal time-of-flight mass spectrometer is a bipolartime-of-flight mass spectrometer.
 16. The apparatus of claim 1, whereinsaid gas flow formation region comprises an exit capillary of said ionsource.
 17. The apparatus of claim 16, wherein said exit capillarycomprises individually biasable gas-tight electrodes.
 18. The apparatusof claim 17, wherein said wherein at least one electrode of said exitcapillary comprises a surface coated with dielectric films.
 19. Theapparatus of claim 1, wherein said at least one sectionedradio-frequency multipole ion guide comprise a sectioned radio-frequencyoctopole ion guide followed by a sectioned radio frequency quadrupoleion guide.
 20. The apparatus of claim 1, wherein said ion sourcecomprises an ion mobility cell.
 21. The apparatus of claim 1, furthercomprising an electron source fluidly coupled to said exit orifice andto said detector.
 22. The apparatus of claim 21, wherein said electronsource is a pulsed electron source, a continuous electron source, or acombination thereof.
 23. The apparatus of claim 1, further comprising amirror assembly between said exit orifice and said detector.
 24. Theapparatus of claim 23, wherein said mirror assembly comprises aparabolic mirror or a cylindrical mirror; and, a flat mirror.
 25. Amethod of analyzing gaseous ions, neutral species or mixtures of ionsand neutral species comprising: introducing said ions and/or neutralspecies into a gas flow formation region to form a gas flow of said ionsand/or neutral species, said gas flow formation region comprising asectioned capillary having at least two electrodes; introducing said gasflow of ions and/or neutral species into at least one sectionedradio-frequency multipole ion guide, said sectioned ion guide comprisinga plurality of electrically isolated electrode sections; applyingvoltage selected from the group consisting of DC voltages, AC voltage,RF voltages, and any combination thereof, to one or more sections ofsaid at least one sectioned radio-frequency multipole ion guide;detecting said gas flow of ions and/or neutral species.
 26. The methodof claim 25, further comprising the step of varying one or more of saidvoltage selected from the group consisting of DC voltages, AC voltage,RF voltages, and any combination thereof.
 27. The method of claim 25,wherein said step of applying voltage to said ion guide comprisesproducing a resonant rotating field which extracts specific ions fromsaid gas flow and causes said specific ions to follow a rotational orbitaround the central axis of said multipole ion guide.
 28. The method ofclaim 27, wherein resonant rotating field voltages are formed byapplying an alternating current harmonic voltage with a phase shift ofπ/2 between adjacent rods within said radio-frequency multipole ionguide.
 29. The method of claim 27, wherein said step of applying voltageto said ion guide further comprises producing a gradient DC field whichtraps said ions following a rotational orbit in a region of the ionguide.
 30. The method of claim 29, wherein said step of applying voltageextracts and traps ions of one or more m/z values and ion mobilitycross-sections.
 31. The method of claim 27, wherein said resonantrotating field voltage is a graded resonant rotating field voltage. 32.The method of claim 25, wherein said step of applying voltage to saidion guide comprises holding specific ions in said gas flow.
 33. Themethod of claim 25, wherein said step of introducing said ions and/orneutral species into a gas flow formation region comprises introducingsaid ions and/or neutral species into a supersonic gas flow.
 34. Themethod of claim 33, wherein said step of applying voltage to said ionguide comprises trapping ions in said supersonic gas flow.
 35. Themethod of claim 34, wherein said step of introducing said ions and/orneutral species into a gas flow formation region comprises adding anadmixture gas.
 36. The method of claim 35, wherein said admixture gascomprises Ar and/or Xe.
 37. The method of claim 35, wherein said step ofadding an admixture gas fragments said trapped ions to create daughterions of said trapped ions.
 38. The method of claim 37, wherein said stepof applying voltage selected from the group consisting of DC voltages,AC voltage, RF voltages, and any combination thereof, to one or moresections of said at least one sectioned radio-frequency multipole ionguide extracts daughter ions from said gas stream.
 39. The method ofclaim 38, further comprising varying said voltage selected from thegroup consisting of DC voltages, AC voltage, RF voltages, and anycombination thereof to reintroduce said daughter ions into said gasstream.
 40. The method of claim 25, further comprising applying a DCvoltage between the last electrode of said sectioned capillary and thefirst electrode section of said radio-frequency multipole ion guide. 41.The method of claim 40, wherein said DC voltage between the lastelectrode of said sectioned capillary and the first electrode section ofsaid radio-frequency multipole ion guide is applied when a desired ionis present in a region between the sectioned capillary and theradio-frequency multipole ion guide.
 42. The method of claim 40, furthercomprising the step of removing said DC voltage between the lastelectrode of said sectioned capillary and the first electrode section ofsaid radio-frequency multipole ion guide.
 43. The method of claim 42,wherein said steps of applying and removing said DC voltage between thelast electrode of said sectioned capillary and the first electrodesection of said radio-frequency multipole ion guide are repeated one ormore times.
 44. The method of claim 38, further comprising the step ofremoving said voltage selected from the group consisting of DC voltages,AC voltage, RF voltages, and any combination thereof, to one or moresections of said at least one sectioned radio-frequency multipole ionguide.
 45. The method of claim 25, further comprising the steps ofapplying a decreasing electric field in the direction of the gas flow insaid radio-frequency multipole ion guide and measuring the mobilitycross-section of said ions.
 46. The method of claim 25, wherein saidstep of detecting comprises detecting with a mass spectrometer.
 47. Themethod of claim 46, wherein said mass spectrometer is an orthogonaltime-of-flight mass spectrometer.
 48. The method of claim 47, whereinsaid orthogonal time-of-flight mass spectrometer comprises a positionsensitive multi-anode detector.
 49. The method of claim 47, wherein saidorthogonal time-of-flight mass spectrometer is a bipolar time-of-flightmass spectrometer.
 50. The method of claim 25, further comprising thestep of passing said gas flow of ions and/or neutral species into amirror assembly.
 51. The method of claim 50, wherein said mirrorassembly comprises a parabolic mirror or a cylindrical mirror; and, aflat mirror.
 52. The method of claim 25, further comprising the step ofimpacting said gas flow of ions and/or neutral species with electronsfrom an electron source or with photons from a laser source.